Plus end-directed microtubule motor required for chromosome congression

ABSTRACT

The invention provides isolated nucleic acid and amino acid sequences of  Xenopus  CENP-E (XCENP-E), antibodies to XCENP-E, methods of screening for CENP-E modulators using biologically active CENP-E, and kits for screening for CENP-E modulators.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Ser. No. 60/058,645, filedSep. 11, 1997, herein incorporated by reference.

FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with Government support under Grant Nos. GM35252and GM 29513, awarded by the National Institutes of Health. TheGovernment has certain rights in this invention.

FIELD OF THE INVENTION

The invention provides isolated nucleic acid and amino acid sequences ofXenopus CENP-E (XCENP-E), antibodies to XCENP-E, methods of screeningfor CENP-E modulators using biologically active CENP-E, and kits forscreening for CENP-E modulators.

BACKGROUND OF THE INVENTION

Segregation of genetic material during mitosis is mediated by themicrotubules of the mitotic spindle (see, e.g., McIntosh, inMicrotubules, pp. 413-434 (Hyams & Lloyd, eds., 1994). During mitosis,chromosomes are dynamically attached to spindle microtubules via thekinetochore, which is a structure located at the centromere of thechromosome. Kinetochores are involved in coordinating chromosomemovement via microtubule assembly and disassembly. The kinetochore andits component proteins thus play an important role in the dynamics ofmitosis.

Spindle microtubules have a defined polarity, with their slow-growing,“minus” ends anchored at or near the spindle pole, and their dynamic,fast-growing “plus” ends interacting with chromosomes (McIntosh, et al.,J. Cell Biol. 98:525-533 (1984)). During prometaphase, chromosomesestablish interactions with the fast-growing plus ends of microtubulesvia the kinetochore. Chromosomes then undergo a series ofmicrotubule-dependent movements, culminating in alignment at themetaphase plate, equidistant from the two spindle poles. This process iscalled “congression.” However, the molecular mechanisms underlyingchromosome congression are poorly understood (see, e.g., Rieder, et al.,J. Cell Biol. 124:223-33 (1994)). A major question has been whether anykinetochore-associated microtubule motors play an important role incongression.

The two predominant and opposing forces are currently thought to beresponsible for chromosome movement during congression: (1) ananti-poleward polar force associated with regions of high microtubuledensity near the spindle poles, and (2) a poleward force generated atthe kinetochore (Khodjakov, et al., J. Cell Biol. 135:315-327 (1996);Waters, et al., J. Cell Sci. 109:2823-2831 (1996); reviewed in Rieder,et al., Int. Rev. Cytol. 79:1-57 (1982); Mitchison, et al., Annu. Rev.Cell Biol. 4:527-49 (1988); Rieder, et al., J. Cell Biol. 124:223-33(1994)).

Studies in vitro have demonstrated the presence of both plus and minusend-directed microtubule motor activities on kinetochores that may beresponsible for these chromosome movements (Mitchison, et al., J. CellBiol. 101:766-77 (1985); Hyman, et al., Nature 351:206-211 (1991)). Theoutstanding issue, however, has been the identity of the molecules atthe kinetochore which act as motors and generate the force forchromosome movement.

In general, both genetic and biochemical approaches have demonstratedcrucial roles for microtubule motors in spindle assembly, spindle poleseparation, and regulation of spindle microtubule dynamics. These motorsinclude Eg5, CHO1/MKlp1, ncd, cut7, bimC, CIN8, KIP1, KAR3, Xklp2,XKCM1, and XCTK2 (Sawin, et al., Nature 359:540-543 (1992); Blangy, etal., Cell 83:1159-1169 (1995); Sawin, et al., J. Cell Biol. 112:925-940(1991); Nislow, et al., J. Cell Biol. 111:511-522 (1990); Endow, et al.,J. Cell Sci. 107:859-867 (1994); Hagan, et al., Nature 347:563-566(1990); Hagan, et al., Nature 356:74-76 (1992); Enos, et al., Cell60:1019-1027 (1990); Hoyt, et al., J. Cell Biol. 118:109-120; Roof, etal., J. Cell Biol. 118:95-108 (1992); Saunders, et al., Cell 70:451-458(1992), Boleti, et al., J. Cell. Biol. 125:1303-1312; Walczak, et al.,Cell 84:37-47 (1996); Walczak, et al., J. Cell Biol. 136:859-70 (1997)).Two kinesin superfamily members, Xenopus Xklp1 and Drosophila nodlocalize to chromosome arms. With the exception of these twochromatin-associated motors, which are thought to mediate polar ejectionforces, none of these other proteins have been implicated directly incongression or in chromosome movement during other phases of mitosis(Theurkauf, et al., J. Cell Biol. 116:1167-1180 (1992); Afshar, et al.,Cell 81:129, Cell 81:128-138 (1995); Vernos, et al., Trends in CellBiol. 5:297-301 (1995)).

A candidate for powering chromosome movement in mitosis iscentromere-associated protein-E (CENP-E), a member of the kinesinsuperfamily of microtubule motor proteins. Human CENP-E has been clonedand is an integral component of the kinetochore (Yen, et al., Nature359:536-539 (1992); Yao, et al., The microtubule motor CENP-E is anintegral component of kinetochore corona fibers that link centromeres tospindle microtubules (manuscript)). CENP-E localizes to kinetochoresthroughout all phases of mitotic chromosome movement (early prometaphasethrough anaphase A) (Yen, et al., Nature 359:536-539 (1992); Brown, etal., J. Cell. Biol. 125:1303-1312 (1994); Lombillo, et al., J. CellBiol. 128:107-115 (1995)).

Previous efforts have suggested a role for CENP-E in mitosis.Microinjection of a monoclonal antibody directed against CENP-E intocultured human cells delays anaphase onset (Yen, et al., EMBO J.10:1245-1254 (1991)). Anti-CENP-E antibody injection into maturing mouseoocytes induces arrest at the first reductional division of meiosis(Duesbery, et al., Proc. Natl. Acad. Sci. USA (in press, 1997)).Antibodies against CENP-E block microtubule depolymerization-dependentminus end-directed movement of purified chromosomes in vitro (Lombillo,et al., J. Cell Biol. 128:107-115 (1995)).

However, these experiments have not demonstrated the precise role ofCENP-E in mitosis, nor have they shown the activity of CENP-E, inparticular any motor activity. Recently, CENP-E was reported to beassociated with minus end-directed microtubule motor activity, raisingthe possibility that CENP-E might be responsible for polewardkinetochore movements (Thrower, et al., EMBO J. 14:918-926 (1995)).However, biologically active CENP-E has never been isolated, neitherfrom naturally occurring nor recombinant sources.

SUMMARY OF THE INVENTION

The present invention provides for the first time biologically activeCENP-E and surprisingly demonstrates, contrary to previous reports, thatCENP-E is a motor that powers chromosome movement toward microtubuleplus ends. Using immunodepletion and antibody addition to Xenopus eggextracts, the present invention further demonstrates that CENP-E playsan essential role in congression. The present invention also providesfor the first time the nucleotide and amino acid sequence of isolatedXenopus CENP-E.

In one aspect, the invention provides an isolated, biologically activeCENP-E protein, wherein the CENP-E protein has the following properties:(i) at least one activity selected from the group consisting of plusend-directed microtubule motor activity, ATPase activity, andmicrotubule binding activity; and (ii) the ability to specifically bindto polyclonal antibodies generated against CENP-E. In one embodiment,the CENP-E protein has an average molecular weight of about 300-350 kDa.

In one embodiment, the CENP-E protein has an amino acid sequence havingat least 34%, or alternatively at least 45%, or alternatively at least55% sequence identity with a XCENP-E motor domain of SEQ ID NO:1.Alternatively, CENP-E has at least 60%, 65% or 70% sequence identitywith a XCENP-E motor domain of SEQ ID NO:1. In an alternativeembodiment, the CENP-E has 70%, or alternatively 75%, or alternatively80%, or alternatively 85%, or alternatively 90% or alternatively 95%amino acid sequence identity to a Xenopus CENP-E core motor domain asmeasured using a sequence comparison algorithm. In an alternativeembodiment, the CENP-E protein has an amino acid sequence of SEQ IDNO:1.

In another embodiment provided herein, the CENP-E protein is encoded bya nucleic acid sequence having at least 70% sequence identity with SEQID NO:2. In another aspect of the present invention, the CENP-E proteinis encoded by a nucleic acid which hybridizes under high stringency to anucleic acid having a sequence complementary to that of SEQ ID NO:2.

In one embodiment, the CENP-E protein is from a human. In alternativeembodiments provided herein, the CENP-E protein is from fungus, insects,or plants.

In an alternative embodiment provided herein, the CENP-E proteinspecifically binds to antibodies generated against Xenopus CENP-E(XCENP-E). In this embodiment, the CENP-E protein has an amino acidsequence having greater than 70%, or alternatively 75% sequence identitywith a XCENP-E motor domain of SEQ ID NO:1. In another embodiment, theCENP-E protein has an amino acid sequence of a XCENP-E motor domain ofSEQ ID NO:1.

In the embodiments wherein the CENP-E is biologically active asdescribed herein, the amino acid sequence can have 74% or less sequenceidentity with the motor domain of SEQ ID NO:1.

Also provided herein is an isolated nucleic acid sequence encoding aCENP-E gene product, said sequence encoding a protein having a coremotor domain that has greater than 70% or alternatively 75% amino acidsequence identity to a Xenopus CENP-E (XCENP-E) core motor domain asmeasured using a sequence comparison algorithm, and specifically bindingto antibodies raised against CENP-E. In one embodiment, the sequence hasa nucleotide sequence of SEQ ID NO:2. The sequence comparison algorithmcan be PILEUP.

In another aspect of the invention, an antibody which specifically bindsto CENP-E is provided.

Also provided herein is a method for identifying a candidate agent as acompound which modulates CENP-E activity. The method comprising thesteps of determining CENP-E activity in the presence of a candidateagent at a control concentration. The CENP-E activity is selected fromthe group consisting of plus end-directed microtubule motor activity,ATPase activity and microtubule binding activity. The method furthercomprises the steps of determining said CENP-E activity in the presencethe candidate agent at a test concentration, wherein a change inactivity between the test concentration and the control concentration ofsaid candidate agent indicates the identification of a compound whichmodulates CENP-E activity. The method can further comprise the step ofisolating biologically active CENP-E from a cell sample.

The compound to be identified can be a lead therapeutic, bioagriculturalcompound or diagnostic. Preferably the compound is an antibody whichspecifically binds CENP-E. In one embodiment the method furthercomprises the step of modifying the antibody to be a humanized antibody.In one embodiment, the method is performed in a plurality such that manycandidate agents are screened simultaneously.

The invention also includes kits for screening for modulators of CENP-E.The kit includes a container holding biologically active CENP-E andinstructions for assaying for CENP-E activity, wherein the CENP-Eactivity is plus end-directed microtubule motor activity or ATPaseactivity.

The invention also provides a method of producing a biologically activeCENP-E polypeptide. The method includes the steps of transforming a cellwith a vector comprising the nucleic acid sequence encoding the motordomain of CENP-E; expressing said nucleic acid to produce a geneproduct; purifying said gene product; and identifying ATPase activity orplus-end directed microtubule activity of said gene product.

In another aspect of the invention, a method of moving microtubules in aplus ended direction is provided wherein microtubules are contacted withbiologically active CENP-E.

In one embodiment the CENP-E is provided in gene form to a cellcomprising microtubules.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A-C: Identification of Xenopus CENP-E

FIG. 1A: Structural comparison of Xenopus and human CENP-E. Hatchedregions represent regions predicted to form a-helical coiled coils(Lupas, et al., Science 252, 1162-1164 (1991)). Within the N-terminalglobular domains of both hCENP-E and XCENP-E there is a domain of ˜324amino acids corresponding to the kinesin like motor domain. Within these324 amino acids XCENP-E and hCENP-E are 74% identical. One cDNA cloneencoded a protein with a 9 amino acid insertion relative to the othercDNAs isolated (see Example I and methods). The position of thisinsertion is marked by the arrowhead. XCENP-E contains a putativenuclear localization signal (NLS) at the C-terminal end of the roddomain not present in hCENP-E.

FIG. 1B: XCENP-E fusion proteins used for polyclonal antibodyproduction.

FIG. 1C: Deduced amino acid sequence of Xenopus CENP-E. cDNA sequencewas compiled from 6 overlapping cDNA clones. Residues identical inhCENP-E and XCENP-E are shaded. The boxed region at the amino-terminusof the sequence is that portion of XCENP-E containing the motor domainand used to assay motility in vitro. The boxed sequence at theC-terminus is that portion of XCENP-E designated as the tail. Theunderlined sequence NSREHSINA (SEQ ID NO:3) at position 599 is the 9amino acid relative insertion encoded by one of the cDNAs isolated (seeFIG. 1A). The putative NLS, RKKTK (SEQ ID NO:4), immediately adjacent tothe boxed tail domain is underlined.

FIG. 2A-B: XCENP-E is a Plus End-Directed Microtubule Motor

FIG. 2A: Expression of recombinant XCENP-E in E. coli. XCENP-E aminoacid residues 1-473 of XCENP-E were fused at the C-terminus to a c-mycepitope followed by a hexahistidine tag, expressed in E. coli, andpurified over Ni-NTA-agarose resin. Coomassie stain of XCENP-E fusionprotein used for motility (lane 1), immunoblot of XCENP-E fusion proteinprobed with α-myc monoclonal antibody (lane 2). Arrowheads indicateXCENP-E fusion protein.

FIG. 2B: XCENP-E Motility Assay. Microtubules marked near their minusends with brightly fluorescent seeds were added with ATP to a flowchamber containing purified XCENP-E fusion protein tethered to thecoverslip with α-myc monoclonal antibody. Gliding of microtubules wasmonitored by time-lapse digital fluorescence microscopy. Selected timepoints from one time lapse series, spaced 90 seconds apart arepresented. As reference points, the positions of the plus ends ofmicrotubules numbered 1, 2, and 3 at the start of continual gliding aremarked with solid white dots, and the position of a stationarymicrotubule end is marked with an arrowhead. The bright seed ofmicrotubule 3 enters the plane of focus at 1.5 minutes, and glides 13.6μM downward with the bright seed leading over the following 3 minutes.Microtubule 2 moves continually during the first three minutes, afterwhich point it detaches and reattaches further toward the bottom of theframe. Microtubule 1 glides minus-end leading throughout the entire timecourse. The average microtubule velocity of all microtubules was 5.1μm/min±1.7 (n=49). Of those, 33 microtubules were unambiguously polaritymarked, and all glided with their bright seeds leading. Scalebar is 5μm.

DETAILED DESCRIPTION OF THE INVENTION I. Introduction

The present invention provides for the first time biologically activeCENP-E and demonstrates that CENP-E has a plus end-directed microtubulemotor activity. Biologically active CENP-E was used to show that thekinesin-like motor domain of CENP-E powers chromosome movement towardmicrotubule plus ends. Finally, quantitative removal of Xenopus CENP-E(“XCENP-E”) from Xenopus egg extracts normally capable of assemblingmitotic spindles in vitro impairs congression of chromosomes to themetaphase plate. Together, these findings demonstrate that CENP-Eplus-end directed microtubule motor activity in vivo is essential forcongression during mitosis.

Functionally, CENP-E is localized in the kinetochores of condensedchromosomes in mitotic cells and has a plus-end directed microtubulemotor activity that is ATP dependent (see, e.g., Example II, where ATPor another nucleotide triphosphate is included in the motility assay formotor activity). This activity is responsible for chromosome movementduring mitosis. Structurally, the full length nucleotide sequence ofXCENP-E (SEQ ID NO:2) encodes a protein of 2954 amino acids with apredicted molecular mass of 340 kDa (SEQ ID NO:1, FIG. 1C). XCENP-E is amember of the kinesin superfamily of motor proteins as evidenced by thesequence of its motor domain. The predicted structure of XCENP-Econsists of a 500 amino acid globular amino-terminal domain containing akinesin-like microtubule motor domain linked to a globular tail domainby a region predicted to form a long, discontinuous α-helical coiledcoil (Lupas, et al., Science 252, 1162-1164 (1991); Berger, et al.,Proc. Natl. Acad. Sci. USA 92:8259-8263 (1995)) (FIG. 1A). Within thecore of the motor domain (residues 1-324) XCENP-E and human CENP-E(“hCENP-E”) share 74% identity (Moore, et al., Bioessays 18:207-219(1996)). Outside the amino-terminal domain lie three additional regionswhich share greater than 25% identity with hCENP-E, but not with otherkinesin-like proteins (FIG. 1). CENP-E is found in Xenopus, mammaliancells, and is predicted to exist in some fungi and perhaps Drosophila.

The isolation of biologically active CENP-E for the first time providesa means for assaying for enhancers or inhibitors (i.e., modulators) ofthis essential mitotic protein. Biologically active CENP-E is useful fortesting for enhancers or inhibitors using in vitro assays such asmicrotubule gliding assays (see, e.g., Example II) or ATPase assays(Kodama et al., J. Biochem. 99: 1465-1472 (1986); Stewart et al., Proc.Nat'l Acad. Sci. USA 90: 5209-5213 (1993); Sakowicz et al., Science280:292-295 (1998)). For example, inhibitors identified usingbiologically active CENP-E can be used therapeutically to treat diseasesof proliferating cells, including, e.g., cancers, hyperplasias,restenosis, cardiac hypertrophy, immune disorders, and inflammation.CENP-E also provides a convenient diagnostic marker for dividing cells.Antibodies or other probes for CENP-E can be used in vitro to identifycells that are entering mitosis. Inhibitors of CENP-E can also be usedin vitro to synchronize cells just prior to entry into mitosis for usein cell culture.

II. Definitions

As used herein, the following terms have the meanings ascribed to themunless specified otherwise.

The terms “isolated” “purified” or “biologically pure” refer to materialthat is substantially or essentially free from components which normallyaccompany it as found in its native state. Purity and homogeneity aretypically determined using analytical chemistry techniques such aspolyacrylamide gel electrophoresis or high performance liquidchromatography. A protein that is the predominant species present in apreparation is substantially purified. In particular, an isolatedXCENP-E nucleic acid is separated from open reading frames which flankthe XCENP-E gene and encode proteins other than XCENP-E. The term“purified” denotes that a nucleic acid or protein gives rise toessentially one band in an electrophoretic gel. Particularly, it meansthat the nucleic acid or protein is at least 85% pure, more preferablyat least 95% pure, and most preferably at least 99% pure.

The term “nucleic acid” refers to deoxyribonucleotides orribonucleotides and polymers thereof in either single- ordouble-stranded form. Unless specifically limited, the term encompassesnucleic acids containing known analogues of natural nucleotides whichhave similar binding properties as the reference nucleic acid and aremetabolized in a manner similar to naturally occurring nucleotides.Unless otherwise indicated, a particular nucleic acid sequence alsoimplicitly encompasses conservatively modified variants thereof (e.g.,degenerate codon substitutions) and complementary sequences and as wellas the sequence explicitly indicated. Specifically, degenerate codonsubstitutions may be achieved by generating sequences in which the thirdposition of one or more selected (or all) codons is substituted withmixed-base and/or deoxyinosine residues (Batzer et al., Nucleic AcidRes. 19:5081 (1991); Ohtsuka et al., J. Biol. Chem. 260:2605-2608(1985); Cassol et al., 1992; Rossolini et al., Mol. Cell. Probes 8:91-98(1994)). The term nucleic acid is used interchangeably with gene, cDNA,and mRNA encoded by a gene.

The terms “polypeptide,” “peptide” and “protein” are usedinterchangeably herein to refer to a polymer of amino acid residues. Theterms apply to amino acid polymers in which one or more amino acidresidue is an artificial chemical analogue of a corresponding naturallyoccurring amino acid, as well as to naturally occurring amino acidpolymers. A CENP-E polypeptide comprises a polypeptide demonstrated tohave at least ATPase activity or plus end-directed microtubule motoractivity and that binds to an antibody generated against CENP-E.

A “label” is a composition detectable by spectroscopic, photochemical,biochemical, immunochemical, or chemical means. For example, usefullabels include ³²P, fluorescent dyes, electron-dense reagents, enzymes(e.g., as commonly used in an ELISA), biotin, dioxigenin, or haptens andproteins for which antisera or monoclonal antibodies are available(e.g., the peptide of SEQ ID NO:1 can be made detectable, e.g., byincorporating a radio-label into the peptide, and used to detectantibodies specifically reactive with the peptide).

As used herein a “nucleic acid probe or oligonucleotide” is defined as anucleic acid capable of binding to a target nucleic acid ofcomplementary sequence through one or more types of chemical bonds,usually through complementary base pairing, usually through hydrogenbond formation. As used herein, a probe may include natural (i.e., A, G,C, or T) or modified bases (7-deazaguanosine, inosine, etc.). Inaddition, the bases in a probe may be joined by a linkage other than aphosphodiester bond, so long as it does not interfere withhybridization. Thus, for example, probes may be peptide nucleic acids inwhich the constituent bases are joined by peptide bonds rather thanphosphodiester linkages. It will be understood by one of skill in theart that probes may bind target sequences lacking completecomplementarity with the probe sequence depending upon the stringency ofthe hybridization conditions. The probes are preferably directly labeledas with isotopes, chromophores, lumiphores, chromogens, or indirectlylabeled such as with biotin to which a streptavidin complex may laterbind. By assaying for the presence or absence of the probe, one candetect the presence or absence of the select sequence or subsequence.

A “labeled nucleic acid probe or oligonucleotide” is one that is bound,either covalently, through a linker, or through ionic, van der Waals orhydrogen bonds to a label such that the presence of the probe may bedetected by detecting the presence of the label bound to the probe.

“Amplification” primers are oligonucleotides comprising either naturalor analogue nucleotides that can serve as the basis for theamplification of a select nucleic acid sequence. They include, e.g.,polymerase chain reaction primers and ligase chain reactionoligonucleotides.

The term “recombinant” when used with reference to a cell, or nucleicacid, or vector, indicates that the cell, or nucleic acid, or vector,has been modified by the introduction of a heterologous nucleic acid orthe alteration of a native nucleic acid, or that the cell is derivedfrom a cell so modified. Thus, for example, recombinant cells expressgenes that are not found within the native (non-recombinant) form of thecell or express native genes that are otherwise abnormally expressed,under expressed or not expressed at all.

The terms “identical” or percent “identity,” in the context of two ormore nucleic acids or polypeptide sequences, refer to two or moresequences or subsequences that are the same or have a specifiedpercentage of amino acid residues or nucleotides that are the same, whencompared and aligned for maximum correspondence over a comparisonwindow, as measured using one of the following sequence comparisonalgorithms or by manual alignment and visual inspection. This definitionalso refers to the complement of a test sequence, which has a designatedpercent sequence or subsequence complementarity when the test sequencehas a designated or substantial identity to a reference sequence. Forexample, a designated amino acid percent identity of 70% refers tosequences or subsequences that have at least about 70% amino acididentity when aligned for maximum correspondence over a comparisonwindow as measured using one of the following sequence comparisonalgorithms or by manual alignment and visual inspection. Preferably, thepercent identity exists over a region of the sequence that is at leastabout 25 amino acids in length, more preferably over a region that is 50or 100 amino acids in length.

When percentage of sequence identity is used in reference to proteins orpeptides, it is recognized that residue positions that are not identicaloften differ by conservative amino acid substitutions, where amino acidsresidues are substituted for other amino acid residues with similarchemical properties (e.g., charge or hydrophobicity) and therefore donot change the functional properties of the molecule. Where sequencesdiffer in conservative substitutions, the percent sequence identity maybe adjusted upwards to correct for the conservative nature of thesubstitution. Means for making this adjustment are well known to thoseof skill in the art. Typically this involves scoring a conservativesubstitution as a partial rather than a full mismatch, therebyincreasing the percentage sequence identity. Thus, for example, where anidentical amino acid is given a score of 1 and a non-conservativesubstitution is given a score of zero, a conservative substitution isgiven a score between zero and 1. The scoring of conservativesubstitutions is calculated according to, e.g., the algorithm of Meyers& Miller, Computer Applic. Biol. Sci. 4:11-17 (1988) e.g., asimplemented in the program PC/GENE (Intelligenetics, Mountain View,Calif., USA).

For sequence comparison, typically one sequence acts as a referencesequence, to which test sequences are compared. When using a sequencecomparison algorithm, test and reference sequences are entered into acomputer, subsequence coordinates are designated, if necessary, andsequence algorithm program parameters are designated. Default programparameters can be used, or alternative parameters can be designated. Thesequence comparison algorithm then calculates the percent sequenceidentity for the test sequence(s) relative to the reference sequence,based on the designated or default program parameters.

A “comparison window”, as used herein, includes reference to a segmentof any one of the number of contiguous positions selected from the groupconsisting of from 25 to 600, usually about 50 to about 200, moreusually about 100 to about 150 in which a sequence may be compared to areference sequence of the same number of contiguous positions after thetwo sequences are optimally aligned. Methods of alignment of sequencesfor comparison are well-known in the art. Optimal alignment of sequencesfor comparison can be conducted, e.g., by the local homology algorithmof Smith & Waterman, Adv. Appl. Math. 2:482 (1981), by the homologyalignment algorithm of Needleman & Wunsch, J. Mol. Biol. 48:443 (1970),by the search for similarity method of Pearson & Lipman, Proc. Nat'l.Acad. Sci. USA 85:2444 (1988), by computerized implementations of thesealgorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin GeneticsSoftware Package, Genetics Computer Group, 575 Science Dr., Madison,Wis.), or by manual alignment and visual inspection (see, e.g., Ausubelet al., supra).

One example of a useful algorithm is PILEUP. PILEUP creates a multiplesequence alignment from a group of related sequences using progressive,pairwise alignments. It can also plot a tree showing the clusteringrelationships used to create the alignment. PILEUP uses a simplificationof the progressive alignment method of Feng & Doolittle, J. Mol. Evol.35:351-360 (1987). The method used is similar to the method described byHiggins & Sharp, CABIOS 5: 151-153 (1989). The program can align up to300 sequences of a maximum length of 5,000. The multiple alignmentprocedure begins with the pairwise alignment of the two most similarsequences, producing a cluster of two aligned sequences. This clustercan then be aligned to the next most related sequence or cluster ofaligned sequences. Two clusters of sequences can be aligned by a simpleextension of the pairwise alignment of two individual sequences. Thefinal alignment is achieved by a series of progressive, pairwisealignments. The program can also be used to plot a dendogram or treerepresentation of clustering relationships. The program is run bydesignating specific sequences and their amino acid or nucleotidecoordinates for regions of sequence comparison, e.g., the core motorregion of CENP-E. In one example, hCENP-E, XCENP-E and ustilago CENP-Ewere compared to other kinesin superfamily sequences using the followingparameters: default gap weight (3.00), default gap length weight (0.10),and weighted end gaps. The resulting dendogram placed hCENP-E andXCENP-E in one cluster as the most closely related sequences, withustilago CENP-E in the next most closely related cluster.

Another example of algorithm that is suitable for determining percentsequence identity (i.e., substantial similarity or identity) is theBLAST algorithm, which is described in Altschul et al., J. Mol. Biol.215:403-410 (1990). Software for performing BLAST analyses is publiclyavailable through the National Center for Biotechnology Information(http://www.ncbi.nlm.nih.gov/). This algorithm involves firstidentifying high scoring sequence pairs (HSPs) by identifying shortwords of length W in the query sequence, which either match or satisfysome positive-valued threshold score T when aligned with a word of thesame length in a database sequence. T is referred to as the neighborhoodword score threshold (Altschul et al, supra). These initial neighborhoodword hits act as seeds for initiating searches to find longer HSPscontaining them. The word hits are then extended in both directionsalong each sequence for as far as the cumulative alignment score can beincreased. Cumulative scores are calculated using, for nucleotidesequences, the parameters M (reward score for a pair of matchingresidues; always >0) and N (penalty score for mismatching residues,always <0). For amino acid sequences, a scoring matrix is used tocalculate the cumulative score. Extension of the word hits in eachdirection are halted when: the cumulative alignment score falls off bythe quantity X from its maximum achieved value; the cumulative scoregoes to zero or below, due to the accumulation of one or morenegative-scoring residue alignments; or the end of either sequence isreached. The BLAST algorithm parameters W, T, and X determine thesensitivity and speed of the alignment. The BLASTN program (fornucleotide sequences) uses as defaults a wordlength (W) of 11, anexpectation (E) of 10, M=5, N=4, and a comparison of both strands. Foramino acid sequences, the BLASTP program uses as default parameters awordlength (W) of 3, an expectation (E) of 10, and the BLOSUM62 scoringmatrix (see Henikoff & Henikoff, Proc. Natl. Acad. Sci. USA 89:10915(1989)).

The BLAST algorithm also performs a statistical analysis of thesimilarity between two sequences (see, e.g., Karlin & Altschul, Proc.Nat'l. Acad. Sci. USA 90:5873-5787 (1993)). One measure of similarityprovided by the BLAST algorithm is the smallest sum probability (P(N)),which provides an indication of the probability by which a match betweentwo nucleotide or amino acid sequences would occur by chance. Forexample, a nucleic acid is considered similar to a reference sequence ifthe smallest sum probability in a comparison of the test nucleic acid tothe reference nucleic acid is less than about 0.1, more preferably lessthan about 0.01, and most preferably less than about 0.001.

An indication that two nucleic acid sequences or polypeptides aresubstantially identical is that the polypeptide encoded by the firstnucleic acid is immunologically cross reactive with the antibodiesraised against the polypeptide encoded by the second nucleic acid, asdescribed below. Thus, a polypeptide is typically substantiallyidentical to a second polypeptide, for example, where the two peptidesdiffer only by conservative substitutions. Another indication that twonucleic acid sequences are substantially identical is that the twomolecules or their complements hybridize to each other under stringentconditions, as described below.

The phrase “hybridizing specifically to” refers to the binding,duplexing, or hybridizing of a molecule only to a particular nucleotidesequence under stringent conditions when that sequence is present in acomplex mixture (e.g., total cellular) DNA or RNA. Stringent conditionsare sequence-dependent and will be different in different circumstances.Longer sequences hybridize specifically at higher temperatures.Generally, stringent conditions are selected to be about 5° C. lowerthan the thermal melting point (T_(m)) for the specific sequence at adefined ionic strength and pH. The T_(m) is the temperature (underdefined ionic strength, pH, and nucleic acid concentration) at which 50%of the probes complementary to the target sequence hybridize to thetarget sequence at equilibrium (as the target sequences are generallypresent in excess, at T_(m), 50% of the probes are occupied atequilibrium). Typically, stringent conditions will be those in which thesalt concentration is less than about 1.0 M sodium ion, typically about0.01 to 1.0 M sodium ion concentration (or other salts) at pH 7.0 to 8.3and the temperature is at least about 30° C. for short probes (e.g., 10to 0.50 nucleotides) and at least about 60° C. for long probes (e.g.,greater than 50 nucleotides). Stringent conditions may also be achievedwith the addition of destabilizing agents such as formamide.

“High stringency conditions”, as defined herein, may be identified bythose that: (1) employ low ionic strength and high temperature forwashing, for example 0.015 M sodium chloride/0.0015 M sodiumcitrate/0.1% sodium dodecyl sulfate at 50° C.; (2) employ duringhybridization a denaturing agent, such as formamide, for example, 50%(v/v) formamide with 0.1% bovine serum albumin/0.1% Ficoll/0.1%polyvinylpyrrolidone/50 mM sodium phosphate buffer at pH 6.5 with 750 mMsodium chloride, 75 mM sodium citrate at 42° C.; or (3) employ 50%formamide, 5×SSC (0.75 M NaCl, 0.075 M sodium citrate), 50 mM sodiumphosphate (pH 6.8), 0.1% sodium pyrophosphate, 5×Denhardt's solution,sonicated salmon sperm DNA (50 μg/ml), 0.1. % SDS, and 10% dextransulfate at 42° C., with washes at 42° C. in 0.2×SSC (sodiumchloride/sodium citrate) and 50% formamide at 55° C., followed by ahigh-stringency wash consisting of 0.1×SSC containing EDTA at 55° C.

“Moderately stringent conditions” may be identified as described bySambrook et al., Molecular Cloning: A Laboratory Manual, New York: ColdSpring Harbor Press, 1989, and include the use of washing solution andhybridization conditions (e.g., temperature, ionic strength and % SDS)less stringent that those described above. An example of moderatelystringent conditions is overnight incubation at 37° C. in a solutioncomprising: 20% formamide, 5×SSC (150 mM NaCl, 15 mM trisodium citrate),50 mM sodium phosphate (pH 7.6), 5×Denhardt's solution, 10% dextransulfate, and 20 mg/mL denatured sheared salmon sperm DNA, followed bywashing the filters in 1×SSC at about 37-50° C. The skilled artisan willrecognize how to adjust the temperature, ionic strength, etc. asnecessary to accommodate factors such as probe length and the like.

The phrase “a sequence encoding a gene product” refers to a nucleic acidthat contains sequence information, e.g., for a structural RNA such asrRNA, a tRNA, the primary amino acid sequence of a specific protein orpeptide, a binding site for a trans-acting regulatory agent, anantisense RNA or a ribozyme. This phrase specifically encompassesdegenerate codons (i.e., different codons which encode a single aminoacid) of the native sequence or sequences which may be introduced toconform with codon preference in a specific host cell.

“Antibody” refers to a polypeptide substantially encoded by animmunoglobulin gene or immunoglobulin genes, or fragments thereof whichspecifically bind and recognize an analyte (antigen). The recognizedimmunoglobulin genes include the kappa, lambda, alpha, gamma, delta,epsilon and mu constant region genes, as well as the myriadimmunoglobulin variable region genes. Light chains are classified aseither kappa or lambda. Heavy chains are classified as gamma, mu, alpha,delta, or epsilon, which in turn define the immunoglobulin classes, IgG,IgM, IgA, IgD and IgE, respectively.

An exemplary immunoglobulin (antibody) structural unit comprises atetramer. Each tetramer is composed of two identical pairs ofpolypeptide chains, each pair having one “light” (about 25 kDa) and one“heavy” chain (about 50-70 kDa). The N-terminus of each chain defines avariable region of about 100 to 110 or more amino acids primarilyresponsible for antigen recognition. The terms variable light chain (V)and variable heavy chain (V_(H)) refer to these light and heavy chainsrespectively.

Antibodies exist e.g., as intact immunoglobulins or as a number of wellcharacterized fragments produced by digestion with various peptidases.Thus, for example, pepsin digests an antibody below the disulfidelinkages in the hinge region to produce F(ab)′_(2,) a dimer of Fab whichitself is a light chain joined to V_(H)—C_(H)1I by a disulfide bond. TheF(ab)′₂ may be reduced under mild conditions to break the disulfidelinkage in the hinge region, thereby converting the F(ab)′₂ dimer intoan Fab′ monomer. The Fab′ monomer is essentially an Fab with part of thehinge region (see Fundamental Immunology (Paul, ed., 3d ed. 1993). Whilevarious antibody fragments are defined in terms of the digestion of anintact antibody, one of skill will appreciate that such fragments may besynthesized de novo either chemically or by using recombinant DNAmethodology. Thus, the term antibody, as used herein, also includesantibody fragments either produced by the modification of wholeantibodies or those synthesized de novo using recombinant DNAmethodologies (e.g., single chain Fv).

An “anti-XCENP-E” antibody is an antibody or antibody fragment thatspecifically binds a polypeptide encoded by the XCENP-E gene, cDNA, or asubsequence thereof.

Humanized forms of non-human antibodies are chimeric immunoglobulins,immunoglobulin chains or fragments thereof (such as Fv, Fab, Fab′,F(ab′)₂ or other antigen-binding subsequences of antibodies) whichcontain minimal sequence derived from non-human immunoglobulin.Humanized antibodies include human immunoglobulins (recipient antibody)in which residues from a complementary determining region (CDR) of therecipient are replaced by residues from a CDR of a non-human species(donor antibody) such as mouse, rat or rabbit having the desiredspecificity, affinity and capacity. In some instances, Fv frameworkresidues of the human immunoglobulin are replaced by correspondingnon-human residues. Humanized antibodies may also comprise residueswhich are found neither in the recipient antibody nor in the importedCDR or framework sequences. In general, the humanized antibody willcomprise substantially all of at least one, and typically two, variabledomains, in which all or substantially all of the CDR regions correspondto those of a non-human immunoglobulin and all or substantially all ofthe FR regions are those of a human immunoglobulin consensus sequence.The humanized antibody optimally also will comprise at least a portionof an immunoglobulin constant region (Fc), typically that of a humanimmunoglobulin (Jones et al., Nature, 321:522-525 (1986); Riechmann etal., Nature, 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol.,2:593-596 (1992)).

Methods for humanizing non-human antibodies are well known in the art.Generally, a humanized antibody has one or more amino acid residuesintroduced into it from a source which is non-human. These non-humanamino acid residues are often referred to as “import” residues, whichare typically taken from an “import” variable domain. Humanization canbe essentially performed following the method of Winter and co-workers(Jones et al., Nature, 321:522-525 (1986); Riechmann et al., Nature,332:323-327 (1988); Verhoeyen et al., Science, 239:1534-1536 (1988)), bysubstituting rodent CDRs or CDR sequences for the correspondingsequences of a human antibody. Accordingly, such “humanized” antibodiesare chimeric antibodies (U.S. Pat. No. 4,816,567), wherein substantiallyless than an intact human variable domain has been substituted by thecorresponding sequence from a non-human species. In practice, humanizedantibodies are typically human antibodies in which some CDR residues andpossibly some FR residues are substituted by residues from analogoussites in rodent antibodies.

Human antibodies can also be produced using various techniques known inthe art, including phage display libraries (Hoogenboom & Winter, J. Mol.Biol., 227:381 (1991); Marks et al., J. Mol. Biol., 222:581 (1991)). Thetechniques of Cole et al. and Boerner et al. are also available for thepreparation of human monoclonal antibodies (Cole et al., MonoclonalAntibodies and Cancer Therapy, Alan R. Liss, p. 77 (1985) and Boerner etal., J. Immunol., 147(1):86-95 (1991)). Similarly, human antibodies canbe made by introducing of human immunoglobulin loci into transgenicanimals, e.g., mice in which the endogenous immunoglobulin genes havebeen partially or completely inactivated. Upon challenge, human antibodyproduction is observed, which closely resembles that seen in humans inall respects, including gene rearrangement, assembly, and antibodyrepertoire. This approach is described, for example, in U.S. Pat. Nos.5,545,807; 5,545,806; 5,569,825; 5,625,126; 5,633,425; 5,661,016, and inthe following scientific publications: Marks et al., Bio/Technology 10,779-783 (1992); Lonberg et al., Nature 368 856-859 (1994); Morrison,Nature 368, 812-13 (1994); Fishwild et al., Nature Biotechnology 14,845-51 (1996); Neuberger, Nature Biotechnology 14, 826 (1996); Lonberg &Huszar, Intern. Rev. Immunol. 13 65-93 (1995).

A “chimeric antibody” is an antibody molecule in which (a) the constantregion, or a portion thereof, is altered, replaced or exchanged so thatthe antigen binding site (variable region) is linked to a constantregion of a different or altered class, effector function and/orspecies, or an entirely different molecule which confers new propertiesto the chimeric antibody, e.g., an enzyme, toxin, hormone, growthfactor, drug, etc.; or (b) the variable region, or a portion thereof, isaltered, replaced or exchanged with a variable region having a differentor altered antigen specificity.

The term “immunoassay” is an assay that uses an antibody to specificallybind an analyte. The immunoassay is characterized by the use of specificbinding properties of a particular antibody to isolate, target, and/orquantify the analyte.

The phrase “specifically (or selectively) binds to an antibody” or“specifically (or selectively) immunoreactive with,” when referring to aprotein or peptide, refers to a binding reaction that is determinativeof the presence of the protein in a heterogeneous population of proteinsand other biologics. Thus, under designated immunoassay conditions, thespecified antibodies bind to a particular protein and do not bind in asignificant amount to other proteins present in the sample. Specificbinding to an antibody under such conditions may require an antibodythat is selected for its specificity for a particular protein. Forexample, antibodies raised to XCENP-E with the amino acid sequenceencoded in SEQ ID NO:1 can be selected to obtain antibodies specificallyimmunoreactive with that protein and not with other proteins, except forpolymorphic variants. A variety of immunoassay formats may be used toselect antibodies specifically immunoreactive with a particular protein.For example, solid-phase ELISA immunoassays are routinely used to selectantibodies specifically immunoreactive with a protein (see, e.g., Harlow& Lane, Antibodies, A Laboratory Manual (1988), for a description ofimmunoassay formats and conditions that can be used to determinespecific immunoreactivity). Typically a specific or selective reactionwill be at least twice background signal or noise and more typicallymore than 10 to 100 times background.

The phrase “plus end-directed microtubule motor activity” refers to theactivity of a motor protein such as CENP-E to power movement toward the“plus” ends of microtubules. Microtubules are conventionally referred toas having plus (fast growing) and minus ends (slow growing). Forexample, microtubules of the mitotic spindle have their slow growing,minus ends anchored at or near the spindle pole, and their dynamic, fastgrowing plus ends interacting with chromosomes and with microtubulesemanating from the opposite pole.

The term “motor domain” or “core motor domain” refers to the domain ofCENP-E that confers the plus end-microtubule motor activity on theprotein.

“CENP-E” refers to centromere-associated protein, which is a member ofthe kinesin superfamily of microtubule motor proteins. CENP-E is anintegral component of the kinetochore structure of the chromosome, whichlinks the chromosome to the spindle microtubules. “XCENP-E” refers toCENP-E isolated from Xenopus. CENP-E has activity such as ATPaseactivity, microtubule binding activity, and plus end-directedmicrotubule motor activity.

“Modulators of CENP-E” refers to modulatory molecules identified usingan in vitro assays for CENP-E activity (e.g., inhibitors and activatorsor enhancers). Such assays include ATPase activity, microtubule gliding,spindle assembly, microtubule depolymerizing activity, and metaphasearrest. Samples or assays that are treated with a at least one candidateagent at a test concentration are compared to control samples having thecandidate agent at a control concentration (which can be zero), toexamine the extent of modulation. Control samples are assigned arelative CENP-E activity value of 100. Modulation of CENP-E is achievedwhen the CENP-E activity value relative to the control is increased ordecreased about at least 10%, 20%, 30%, 40%, 50%, 75%, or preferably, atleast 100%.

“Biologically active” CENP-E refers to CENP-E that has at least oneactivity selected ATPase activity, microtubule binding activity, andplus end-directed microtubule motor activity, as tested in an ATPaseassay, microtubule binding assay, or a microtubule gliding assay.“ATPase activity” refers to the ability of CENP-E to hydrolyze ATP. In apreferred embodiment, CENP-E has plus-end directed microtubule activity.

III. Isolation of the XCENP-E Gene

A. General Recombinant DNA Methods

This invention relies on routine techniques in the field of recombinantgenetics. Basic texts disclosing the general methods of use in thisinvention include Sambrook et al., Molecular Cloning, A LaboratoryManual (2nd ed. 1989); Kriegler, Gene Transfer and Expression: ALaboratory Manual (1990); and Current Protocols in Molecular Biology(Ausubel et al., eds., 1994)).

For nucleic acids, sizes are given in either kilobases (kb) or basepairs (bp). These are estimates derived from agarose or acrylamide gelelectrophoresis, from sequenced nucleic acids, or from published DNAsequences. For proteins, sizes are given in kilodaltons (kDa) or aminoacid residue numbers. Proteins sizes are estimated from gelelectrophoresis, from sequenced proteins, from derived amino acidsequences, or from published protein sequences.

Oligonucleotides that are not commercially available can be chemicallysynthesized according to the solid phase phosphoramidite triester methodfirst described by Beaucage & Caruthers, Tetrahedron Letts.,22:1859-1862 (1981), using an automated synthesizer, as described in VanDevanter et. al., Nucleic Acids Res., 12:6159-6168 (1984). Purificationof oligonucleotides is by either native acrylamide gel electrophoresisor by anion-exchange HPLC as described in Pearson & Reanier, J. Chrom.,255:137-149 (1983).

The sequence of the cloned genes and synthetic oligonucleotides can beverified after cloning using, e.g., the chain termination method forsequencing double-stranded templates of Wallace et al., Gene, 16:21-26,1981.

B. Cloning Methods for the Isolation of Nucleotide Sequences EncodingXCENP-E

In general, the nucleic acid sequences encoding XCENP-E and relatednucleic acid sequence homologues are cloned from cDNA and genomic DNAlibraries or isolated using amplification techniques witholigonucleotide primers. For example, XCENP-E sequences can be isolatedfrom Xenopus DNA libraries by hybridizing with a nucleic acid probe, thesequence of which can be derived from human CENP-E. XCENP-E and XCENP-Ehomologues that are substantially identical to XCENP-E can be isolatedusing XCENP-E nucleic acid probes and oligonucleotides under stringenthybridization conditions, by screening libraries. Alternatively,expression libraries can be used to clone XCENP-E and XCENP-Ehomologues, by detecting expressed homologues immunologically withantisera or purified antibodies made against XCENP-E that also recognizeand selectively bind to the XCENP-E homologue. Finally, amplificationtechniques using primers can be used to amplify and isolate XCENP-E fromDNA or RNA. Amplification techniques using degenerate primers can alsobe used to amplify and isolate XCENP-E homologues. Amplificationtechniques using primers can also be used to isolate a nucleic acidencoding XCENP-E. These primers can be used, e.g., to amplify a probe ofseveral hundred nucleotides, which is then used to screen a library forfull-length XCENP-E. The following primers can be used in such a manner:SEQ ID NO:5 GGGCTGCCCAGGAAGAG and SEQ ID NO:6 GACAGCATTGATCGGCG.Alternatively, the nucleic acid for XCENP-E can be directly amplifiedusing the following primers: SEQ ID NO:7 GAGGGTTCGGCCGCTTA and SEQ IDNO:8 TCTGGGGCCATCCATGC.

Appropriate primers and probes for identifying the gene encoding CENP-Ein other species such as Drosophila and fungi are generated fromcomparisons of the sequences provided herein (SEQ ID NOS:1 and 2). Asdescribed above, antibodies can be used to identify XCENP-E homologues.For example, antibodies made to the motor domain of XCENP-E, the taildomain of XCENP-E, or to the whole protein are useful for identifyingXCENP-E homologues (see Example section, below).

To make a cDNA library, one should choose a source that is rich in themRNA of choice, e.g., XCENP-E. For example, ovary tissue is enriched forXCENP-E mRNA. The mRNA is then made into cDNA using reversetranscriptase, ligated into a recombinant vector, and transfected into arecombinant host for propagation, screening and cloning. Methods formaking and screening cDNA libraries are well known (see, e.g., Gubler &Hoffman, Gene 25: 263-269 (1983); Sambrook et al., supra; Ausubel etal., supra).

For a genomic library, the DNA is extracted from the tissue and eithermechanically sheared or enzymatically digested to yield fragments ofabout 12-20 kb. The fragments are then separated by gradientcentrifugation from undesired sizes and are constructed in bacteriophagelambda vectors. These vectors and phage are packaged in vitro.Recombinant phage are analyzed by plaque hybridization as described inBenton & Davis, Science 196:180-182 (1977). Colony hybridization iscarried out as generally described in Grunstein et al., Proc. Natl.Acad. Sci. USA., 72:3961-3965 (1975).

An alternative method of isolating XCENP-E nucleic acid and itshomologues combines the use of synthetic oligonucleotide primers andamplification of an RNA or DNA template (see U.S. Pat. Nos. 4,683,195and 4,683,202; PCR Protocols: A Guide to Methods and Applications (Inniset al., eds, 1990)). Methods such as polymerase chain reaction (PCR) andligase chain reaction (LCR) can be used to amplify nucleic acidsequences of CENP-E directly from mRNA, from cDNA, from genomiclibraries or cDNA libraries. Degenerate oligonucleotides can be designedto amplify XCENP-E homologues using the sequences provided herein.Restriction endonuclease sites can be incorporated into the primers.Polymerase chain reaction or other in vitro amplification methods mayalso be useful, for example, to clone nucleic acid sequences that codefor proteins to be expressed, to make nucleic acids to use as probes fordetecting the presence of CENP-E encoding mRNA in physiological samples,for nucleic acid sequencing, or for other purposes. Genes amplified bythe PCR reaction can be purified from agarose gels and cloned into anappropriate vector.

Gene expression of CENP-E can also be analyzed by techniques known inthe art, e.g., reverse transcription and amplification of mRNA,isolation of total RNA or poly A+RNA, northern blotting, dot blotting,in situ hybridization, RNase protection and the like.

Synthetic oligonucleotides can be used to construct recombinant XCENP-Egenes for use as probes or for expression of protein. This method isperformed using a series of overlapping oligonucleotides usually 40-120bp in length, representing both the sense and nonsense strands of thegene. These DNA fragments are then annealed, ligated and cloned.Alternatively, amplification techniques can be used with precise primersto amplify a specific subsequence of the XCENP-E gene. The specificsubsequence is then ligated into an expression vector.

The gene for Xenopus CENP-E is typically cloned into intermediatevectors before transformation into prokaryotic or eukaryotic cells forreplication and/or expression. These intermediate vectors are typicallyprokaryote vectors or shuttle vectors.

C. Expression in Prokaryotes and Eukaryotes

To obtain high level expression of a cloned gene, such as those cDNAsencoding CENP-E, it is important to construct an expression vector thatcontains, at the minimum, a strong promoter to direct transcription, aribosome binding site for translational initiation, and atranscription/translation terminator. Suitable bacterial promoters arewell known in the art and described, e.g., in Sambrook et al. andAusubel et al. Bacterial expression systems for expressing the CENP-Eprotein are available in, e.g., E. coli, Bacillus sp., and Salmonella(Palva et al., Gene 22:229-235 (1983); Mosbach, et al., Nature,302:543-545 (1983). Kits for such expression systems are commerciallyavailable. Eukaryotic expression systems for mammalian cells, yeast, andinsect cells are well known in the art and are also commerciallyavailable. The pET23D expression system (Novagen) is a preferredprokaryotic expression system.

A “promoter” is defined as an array of nucleic acid control sequencesthat direct transcription of a nucleic acid. As used herein, a promoterincludes necessary nucleic acid sequences near the start site oftranscription, such as, in the case of a polymerase II type promoter, aTATA element. A promoter also optionally includes distal enhancer orrepressor elements which can be located as much as several thousand basepairs from the start site of transcription. A “constitutive” promoter isa promoter which is active under most environmental and developmentalconditions. An “inducible” promoter is a promoter which is underenvironmental or developmental regulation. The term “operably linked”refers to a functional linkage between a nucleic acid expression controlsequence (such as a promoter, or array of transcription factor bindingsites) and a second nucleic acid sequence, wherein the expressioncontrol sequence directs transcription of the nucleic acid correspondingto the second sequence.

The term “heterologous” when used with reference to portions of anucleic acid indicates that the nucleic acid comprises two or moresubsequences which are not found in the same relationship to each otherin nature. For instance, the nucleic acid is typically recombinantlyproduced, having two or more sequences from unrelated genes arranged tomake a new functional nucleic acid.

The promoter used to direct expression of a heterologous nucleic aciddepends on the particular application. The promoter is preferablypositioned about the same distance from the heterologous transcriptionstart site as it is from the transcription start site in its naturalsetting. As is known in the art, however, some variation in thisdistance can be accommodated without loss of promoter function.

In addition to the promoter, the expression vector typically contains atranscription unit or expression cassette that contains all theadditional elements required for the expression of the CENP-E encodingDNA in host cells. A typical expression cassette thus contains apromoter operably linked to the DNA sequence encoding CENP-E and signalsrequired for efficient polyadenylation of the transcript, ribosomebinding sites, and translation termination. The DNA sequence encodingthe CENP-E may typically be linked to a cleavable signal peptidesequence to promote secretion of the encoded protein by the transformedcell. Such signal peptides would include, among others, the signalpeptides from tissue plasminogen activator, insulin, and neuron growthfactor, and juvenile hormone esterase of Heliothis virescens. Additionalelements of the cassette may include enhancers and, if genomic DNA isused as the structural gene, introns with functional splice donor andacceptor sites.

In addition to a promoter sequence, the expression cassette should alsocontain a transcription termination region downstream of the structuralgene to provide for efficient termination. The termination region may beobtained from the same gene as the promoter sequence or may be obtainedfrom different genes.

The particular expression vector used to transport the geneticinformation into the cell is not particularly critical. Any of theconventional vectors used for expression in eukaryotic or prokaryoticcells may be used. Standard bacterial expression vectors includeplasmids such as pBR322 based plasmids, pSKF, and fusion expressionsystems such as GST and LacZ. Epitope tags can also be added torecombinant proteins to provide convenient methods of isolation. Onepreferred embodiment of an epitope tag is c-myc.

Expression vectors containing regulatory elements from eukaryoticviruses are typically used in eukaryotic expression vectors, e.g., SV40vectors, papilloma virus vectors, and vectors derived from Epstein Barvirus. Other exemplary eukaryotic vectors include pMSG, pAV009/A⁺,pMTO10/A⁺, pMAMneo-5, baculovirus pDSVE, and any other vector allowingexpression of proteins under the direction of the SV40 early promoter,SV40 later promoter, metallothionein promoter, murine mammary tumorvirus promoter, Rous sarcoma virus promoter, polyhedrin promoter, orother promoters shown effective for expression in eukaryotic cells.

Some expression systems have markers that provide gene amplificationsuch as thymidine kinase, hygromycin B phosphotransferase, anddihydrofolate reductase. Alternatively, high yield expression systemsnot involving gene amplification are also suitable, such as using abaculovirus vector in insect cells, with a CENP-E encoding sequenceunder the direction of the polyhedrin promoter or other strongbaculovirus promoters.

The elements that are typically included in expression vectors alsoinclude a replicon that functions in E. coli, a gene encoding antibioticresistance to permit selection of bacteria that harbor recombinantplasmids, and unique restriction sites in nonessential regions of theplasmid to allow insertion of eukaryotic sequences. The particularantibiotic resistance gene chosen is not critical, any of the manyresistance genes known in the art are suitable. The prokaryoticsequences are preferably chosen such that they do not interfere with thereplication of the DNA in eukaryotic cells, if necessary.

Standard transfection methods are used to produce bacterial, mammalian,yeast or insect cell lines that express large quantities of CENP-Eprotein, which are then purified using standard techniques (see, e.g.,Colley et al., J. Biol. Chem. 264:17619-17622 (1989); Guide to ProteinPurification, in Methods in Enzymology, vol. 182 (Deutscher ed., 1990)).Transformation of eukaryotic and prokaryotic cells are performedaccording to standard techniques (see, e.g., Morrison, J. Bact.,132:349-351 (1977); Clark-Curtiss & Curtiss, Methods in Enzymology,101:347-362 (Wu et al., eds, 1983).

Any of the well known procedures for introducing foreign nucleotidesequences into host cells may be used. These include the use of calciumphosphate transfection, polybrene, protoplast fusion, electroporation,liposomes, microinjection, plasma vectors, viral vectors and any of theother well known methods for introducing cloned genomic DNA, cDNA,synthetic DNA or other foreign genetic material into a host cell (see,e.g., Sambrook et al., supra). It is only necessary that the particulargenetic engineering procedure used be capable of successfullyintroducing at least one gene into the host cell capable of expressingthe CENP-E protein.

After the expression vector is introduced into the cells, thetransfected cells are cultured under conditions favoring expression ofthe CENP-E protein which is recovered from the culture using standardtechniques identified below.

IV. Purification of CENP-E Protein

Either naturally occurring or recombinant CENP-E can be purified for usein functional assays. Naturally occurring CENP-E is purified, e.g., fromXenopus and any other source of an XCENP-E homologue, such as Drosophilaor fungi. Recombinant CENP-E is purified from any suitable expressionsystem.

CENP-E may be purified to substantial purity by standard techniques,including selective precipitation with such substances as ammoniumsulfate; column chromatography, immunopurification methods, and others(see, e.g., Scopes, Protein Purification: Principles and Practice(1982); U.S. Pat. No. 4,673,641; Ausubel et al., supra; and Sambrook etal., supra). A preferred method of purification is use of Ni-NTA agarose(Qiagen).

A number of procedures can be employed when recombinant CENP-E is beingpurified. For example, proteins having established molecular adhesionproperties can be reversible fused to CENP-E. With the appropriateligand, CENP-E can be selectively adsorbed to a purification column andthen freed from the column in a relatively pure form. The fused proteinis then removed by enzymatic activity. Finally CENP-E could be purifiedusing immunoaffinity columns.

A. Purification of CENP-E from Recombinant Bacteria

Recombinant proteins are expressed by transformed bacteria in largeamounts, typically after promoter induction; but expression can beconstitutive. Bacteria are grown according to standard procedures in theart. Because CENP-E is a protein that is difficult to isolate withintact biological activity, preferably fresh bacteria cells are used forisolation of protein. Use of cells that are frozen after growth butprior to lysis typically results in negligible yields of active protein.

Proteins expressed in bacteria may form insoluble aggregates (“inclusionbodies”). Several protocols are suitable for purification of CENP-Einclusion bodies. For example, purification of inclusion bodiestypically involves the extraction, separation and/or purification ofinclusion bodies by disruption of bacterial cells, e.g., by incubationin a buffer of about 100-150 μg/ml lysozyme and 0.1% Nonidet P40, anon-ionic detergent. The cell suspension can be homogenized using aPolytron (Brinkman Instruments, Westbury, N.Y.). Alternatively, thecells can be sonicated on ice. Alternate methods of lysing bacteria areapparent to those of skill in the art (see, e.g., Sambrook et al.,supra; Ausubel et al., supra).

The cell suspension is generally centrifuged and the pellet containingthe inclusion bodies resuspended in buffer that does not dissolve butwashes the inclusion bodies, e.g., 20 mM Tris-HCl (pH 7.2), 1 mM EDTA,150 mM NaCl and 2% Triton-X 100, a non-ionic detergent. It may benecessary to repeat the wash step to remove as much cellular debris aspossible. The remaining pellet of inclusion bodies may be resuspended inan appropriate buffer (e.g., 20 mM sodium phosphate, pH 6.8, 150 mMNaCl). Other appropriate buffers will be apparent to those of skill inthe art.

Following the washing step, the inclusion bodies are solubilized by theaddition of a solvent that is both a strong hydrogen acceptor and astrong hydrogen donor (or a combination of solvents each having one ofthese properties); the proteins that formed the inclusion bodies maythen be renatured by dilution or dialysis with a compatible buffer.Suitable solvents include, but are not limited to urea (from about 4 Mto about 8 M), formamide (at least about 80%, volume/volume basis), andguanidine hydrochloride (from about 4 M to about 8 M). Some solventswhich are capable of solubilizing aggregate-forming proteins, forexample SDS (sodium dodecyl sulfate), 70% formic acid, are inappropriatefor use in this procedure due to the possibility of irreversibledenaturation of the proteins, accompanied by a lack of immunogenicityand/or activity. Although guanidine hydrochloride and similar agents aredenaturants, this denaturation is not irreversible and renaturation mayoccur upon removal (by dialysis, for example) or dilution of thedenaturant, allowing re-formation of immunologically and/or biologicallyactive protein. After solubilization, the protein can be separated fromother bacterial proteins by standard separation techniques.

Alternatively, it is possible to purify CENP-E from bacteria periplasm.Where CENP-E is exported into the periplasm of the bacteria, theperiplasmic fraction of the bacteria can be isolated by cold osmoticshock in addition to other methods known to skill in the art. To isolaterecombinant proteins from the periplasm, the bacterial cells arecentrifuged to form a pellet. The pellet is resuspended in a buffercontaining 20% sucrose. To lyse the cells, the bacteria are centrifugedand the pellet is resuspended in ice-cold 5 mM MgSO₄ and kept in an icebath for approximately 10 minutes. The cell suspension is centrifugedand the supernatant decanted and saved. The recombinant proteins presentin the supernatant can be separated from the host proteins by standardseparation techniques well known to those of skill in the art.

B. Standard Protein Separation Techniques for Purifying CENP-E

Solubility Fractionation

Often as an initial step, particularly if the protein mixture iscomplex, an initial salt fractionation can separate many of the unwantedhost cell proteins (or proteins derived from the cell culture media)from the recombinant protein of interest. The preferred salt is ammoniumsulfate. Ammonium sulfate precipitates proteins by effectively reducingthe amount of water in the protein mixture. Proteins then precipitate onthe basis of their solubility. The more hydrophobic a protein is, themore likely it is to precipitate at lower ammonium sulfateconcentrations. A typical protocol includes adding saturated ammoniumsulfate to a protein solution so that the resultant ammonium sulfateconcentration is between 20-30%. This concentration will precipitate themost hydrophobic of proteins. The precipitate is then discarded (unlessthe protein of interest is hydrophobic) and ammonium sulfate is added tothe supernatant to a concentration known to precipitate the protein ofinterest. The precipitate is then solubilized in buffer and the excesssalt removed if necessary, either through dialysis or diafiltration.Other methods that rely on solubility of proteins, such as cold ethanolprecipitation, are well known to those of skill in the art and can beused to fractionate complex protein mixtures.

Size Differential Filtration

CENP-E has a known molecular weight and this knowledge can be used toisolated it from proteins of greater and lesser size usingultrafiltration through membranes of different pore size (for example,Amicon or Millipore membranes). As a first step, the protein mixture isultrafiltered through a membrane with a pore size that has a lowermolecular weight cut-off than the molecular weight of the protein ofinterest. The retentate of the ultrafiltration is then ultrafilteredagainst a membrane with a molecular cut off greater than the molecularweight of the protein of interest. The recombinant protein will passthrough the membrane into the filtrate. The filtrate can then bechromatographed as described below.

Column Chromatography

CENP-E can also be separated from other proteins on the basis of itssize, net surface charge, hydrophobicity, and affinity for ligands. Inaddition, antibodies raised against proteins can be conjugated to columnmatrices and the proteins immunopurified. All of these methods are wellknown in the art. It will be apparent to one of skill thatchromatographic techniques can be performed at any scale and usingequipment from many different manufacturers (e.g., Pharmacia Biotech).

V. Immunological Detection of CENP-E

In addition to the detection of CENP-E genes and gene expression usingnucleic acid hybridization technology, one can also use immunoassays todetect CENP-E. Immunoassays can be used to qualitatively orquantitatively analyze CENP-E. A general overview of the applicabletechnology can be found in Harlow & Lane, Antibodies: A LaboratoryManual (1988).

A. Antibodies to CENP-E

Methods of producing polyclonal and monoclonal antibodies that reactspecifically with CENP-E are known to those of skill in the art (see,e.g., Coligan, Current Protocols in Immunology (1991); Harlow & Lane,supra; Goding, Monoclonal Antibodies: Principles and Practice (2d ed.1986); and Kohler & Milstein, Nature, 256:495-497 (1975). Suchtechniques include antibody preparation by selection of antibodies fromlibraries of recombinant antibodies in phage or similar vectors, as wellas preparation of polyclonal and monoclonal antibodies by immunizingrabbits or mice (see, e.g., Huse et al., Science 246:1275-1281 (1989);Ward et al., Nature 341:544-546 (1989)).

A number of CENP-E comprising immunogens may be used to produceantibodies specifically reactive with CENP-E. For example, recombinantXCENP-E or a antigenic fragment thereof such as the motor or taildomain, is isolated as described herein. Recombinant protein can beexpressed in eukaryotic or prokaryotic cells as described above, andpurified as generally described above. Recombinant protein is thepreferred immunogen for the production of monoclonal or polyclonalantibodies. Alternatively, a synthetic peptide derived from thesequences disclosed herein and conjugated to a carrier protein can beused an immunogen. Naturally occurring protein may also be used eitherin pure or impure form. The product is then injected into an animalcapable of producing antibodies. Either monoclonal or polyclonalantibodies may be generated, for subsequent use in immunoassays tomeasure the protein.

Methods of production of polyclonal antibodies are known to those ofskill in the art. An inbred strain of mice or rabbits is immunized withthe protein using a standard adjuvant, such as Freund's adjuvant, and astandard immunization protocol. The animal's immune response to theimmunogen preparation is monitored by taking test bleeds and determiningthe titer of reactivity to CENP-E. When appropriately high titers ofantibody to the immunogen are obtained, blood is collected from theanimal and antisera are prepared. Further fractionation of the antiserato enrich for antibodies reactive to the protein can be done if desired(see Harlow & Lane, supra).

Monoclonal antibodies may be obtained by various techniques familiar tothose skilled in the art. Briefly, spleen cells from an animal immunizedwith a desired antigen are immortalized, commonly by fusion with amyeloma cell (see Kohler & Milstein, Eur. J. Immunol. 6:511-519 (1976)).Alternative methods of immortalization include transformation withEpstein Barr Virus, oncogenes, or retroviruses, or other methods wellknown in the art. Colonies arising from single immortalized cells arescreened for production of antibodies of the desired specificity andaffinity for the antigen, and yield of the monoclonal antibodiesproduced by such cells may be enhanced by various techniques, includinginjection into the peritoneal cavity of a vertebrate host.Alternatively, one may isolate DNA sequences which encode a monoclonalantibody or a binding fragment thereof by screening a DNA library fromhuman B cells according to the general protocol outlined by Huse et al.,Science 246:1275-1281 (1989).

Monoclonal antibodies and polyclonal sera are collected and titeredagainst the immunogen protein in an immunoassay, for example, a solidphase immunoassay with the immunogen immobilized on a solid support.Polyclonal antisera with a titer of 10⁴ or greater are selected andtested for their cross reactivity against non-CENP-E proteins or evenother homologous proteins from other organisms, using a competitivebinding immunoassay. Specific polyclonal antisera and monoclonalantibodies will usually bind with a K_(D) of at least about 0.1 mM, moreusually at least about 1 μM, preferably at least about 0.1 μM or better,and most preferably, 0.01 μM or better.

Once CENP-E specific antibodies are available, CENP-E can be detected bya variety of immunoassay methods. For a review of immunological andimmunoassay procedures, see Basic and Clinical Immunology (Stites & Terreds., 7th ed. 1991). Moreover, the immunoassays of the present inventioncan be performed in any of several configurations, which are reviewedextensively in Enzyme Immunoassay (Maggio, ed., 1980); and Harlow &Lane, supra.

B. Immunological Binding Assays

As explained above, CENP-E expression is associated with mitosis. Thus,CENP-E provides a marker with which to examine actively dividing cells,including pathological cells such as cancers or hyperplasias. In apreferred embodiment, CENP-E is detected and/or quantified using any ofa number of well recognized immunological binding assays (see, e.g.,U.S. Pat. Nos. 4,366,241; 4,376,110; 4,517,288; and 4,837,168). For areview of the general immunoassays, see also Methods in Cell BiologyVolume 37: Antibodies in Cell Biology (Asai, ed. 1993); Basic andClinical Immunology (Stites & Terr, eds., 7th ed. 1991). Immunologicalbinding assays (or immunoassays) typically utilize a “capture agent” tospecifically bind to and often immobilize the analyte (in this case theCENP-E or antigenic subsequence thereof). The capture agent is a moietythat specifically binds to the analyte. The antibody (anti-CENP-E) maybe produced by any of a number of means well known to those of skill inthe art and as described above.

Immunoassays also often utilize a labeling agent to specifically bind toand label the binding complex formed by the capture agent and theanalyte. The labeling agent may itself be one of the moieties comprisingthe antibody/analyte complex. Thus, the labeling agent may be a labeledCENP-E polypeptide or a labeled anti-CENP-E antibody. Alternatively, thelabeling agent may be a third moiety, such as another antibody, thatspecifically binds to the antibody/CENP-E complex.

In a preferred embodiment, the labeling agent is a second CENP-E bearinga label. Alternatively, the second antibody may lack a label, but itmay, in turn, be bound by a labeled third antibody specific toantibodies of the species from which the second antibody is derived. Thesecond can be modified with a detectable moiety, such as biotin, towhich a third labeled molecule can specifically bind, such asenzyme-labeled streptavidin.

Other proteins capable of specifically binding immunoglobulin constantregions, such as protein A or protein G may also be used as the labelagent. These proteins are normal constituents of the cell walls ofstreptococcal bacteria. They exhibit a strong non-immunogenic reactivitywith immunoglobulin constant regions from a variety of species (seegenerally Kronval, et al., J. Immunol., 111: 1401-1406 (1973);Akerstrom, et al., J. Immunol., 135: 2589-2542 (1985)).

Throughout the assays, incubation and/or washing steps may be requiredafter each combination of reagents. Incubation steps can vary from about5 seconds to several hours, preferably from about 5 minutes to about 24hours. However, the incubation time will depend upon the assay format,analyte, volume of solution, concentrations, and the like. Usually, theassays will be carried out at ambient temperature, although they can beconducted over a range of temperatures, such as 10° C. to 40° C.

Non-Competitive Assay Formats

Immunoassays for detecting CENP-E in samples may be either competitiveor noncompetitive. Noncompetitive immunoassays are assays in which theamount of captured analyte (in this case the protein) is directlymeasured. In one preferred “sandwich” assay, for example, the captureagent (anti-CENP-E antibodies) can be bound directly to a solidsubstrate on which they are immobilized. These immobilized antibodiesthen capture CENP-E present in the test sample. CENP-E is thusimmobilized is then bound by a labeling agent, such as a second CENP-Eantibody bearing a label. Alternatively, the second antibody may lack alabel, but it may, in turn, be bound by a labeled third antibodyspecific to antibodies of the species from which the second antibody isderived. The second can be modified with a detectable moiety, such asbiotin, to which a third labeled molecule can specifically bind, such asenzyme-labeled streptavidin.

Competitive Assay Formats

In competitive assays, the amount of CENP-E (analyte) present in thesample is measured indirectly by measuring the amount of an added(exogenous) analyte (i.e the CENP-E) displaced (or competed away) from acapture agent (anti-CENP-E antibody) by the analyte present in thesample. In one competitive assay, a known amount of, in this case, theCENP-E is added to the sample and the sample is then contacted with acapture agent, in this case an antibody that specifically binds to theCENP-E. The amount of CENP-E bound to the antibody is inverselyproportional to the concentration of CENP-E present in the sample. In aparticularly preferred embodiment, the antibody is immobilized on asolid substrate. The amount of the CENP-E bound to the antibody may bedetermined either by measuring the amount of CENP-E present in anCENP-E/antibody complex, or alternatively by measuring the amount ofremaining uncomplexed protein. The amount of CENP-E may be detected byproviding a labeled CENP-E molecule.

A hapten inhibition assay is another preferred competitive assay. Inthis assay a known analyte, in this case CENP-E, is immobilized on asolid substrate. A known amount of anti-CENP-E antibody is added to thesample, and the sample is then contacted with the immobilized CENP-E.The amount of anti-CENP-E antibody bound to the immobilized CENP-E isinversely proportional to the amount of CENP-E present in the sample.Again, the amount of immobilized antibody may be detected by detectingeither the immobilized fraction of antibody or the fraction of theantibody that remains in solution. Detection may be direct where theantibody is labeled or indirect by the subsequent addition of a labeledmoiety that specifically binds to the antibody as described above.

Immunoassays in the competitive binding format can be used forcrossreactivity determinations. For example, a protein partially encodedby SEQ ID NO:1 can be immobilized to a solid support. Proteins are addedto the assay that compete with the binding of the antisera to theimmobilized antigen. The ability of the above proteins to compete withthe binding of the antisera to the immobilized protein is compared toCENP-E encoded by SEQ ID NO:1. The percent crossreactivity for the aboveproteins is calculated, using standard calculations. Those antisera withless than 10% crossreactivity with each of the proteins listed above areselected and pooled. The cross-reacting antibodies are optionallyremoved from the pooled antisera by immunoabsorption with the consideredproteins, e.g., distantly related homologues.

The immunoabsorbed and pooled antisera are then used in a competitivebinding immunoassay as described above to compare a second protein,thought to be perhaps the protein of this invention, to the immunogenprotein (i.e., CENP-E of SEQ ID NO:1). In order to make this comparison,the two proteins are each assayed at a wide range of concentrations andthe amount of each protein required to inhibit 50% of the binding of theantisera to the immobilized protein is determined. If the amount of thesecond protein required to inhibit 50% of binding is less than 10 timesthe amount of the protein partially encoded by SEQ ID NO:1 that isrequired to inhibit 50% of binding, then the second protein is said tospecifically bind to the polyclonal antibodies generated to an CENP-Eimmunogen.

Other Assay Formats

Western blot (immunoblot) analysis is used to detect and quantify thepresence of CENP-E in the sample. The technique generally comprisesseparating sample proteins by gel electrophoresis on the basis ofmolecular weight, transferring the separated proteins to a suitablesolid support, (such as a nitrocellulose filter, a nylon filter, orderivatized nylon filter), and incubating the sample with the antibodiesthat specifically bind the CENP-E. The anti-CENP-E antibodiesspecifically bind to the CENP-E on the solid support. These antibodiesmay be directly labeled or alternatively may be subsequently detectedusing labeled antibodies (e.g., labeled sheep anti-mouse antibodies)that specifically bind to the anti-CENP-E antibodies.

Other assay formats include liposome immunoassays (LIA), which useliposomes designed to bind specific molecules (e.g., antibodies) andrelease encapsulated reagents or markers. The released chemicals arethen detected according to standard techniques (see Monroe et al., Amer.Clin. Prod. Rev. 5:3441 (1986)).

Reduction of Non-Specific Binding

One of skill in the art will appreciate that it is often desirable tominimize non-specific binding in immunoassays. Particularly, where theassay involves an antigen or antibody immobilized on a solid substrateit is desirable to minimize the amount of non-specific binding to thesubstrate. Means of reducing such non-specific binding are well known tothose of skill in the art. Typically, this technique involves coatingthe substrate with a proteinaceous composition. In particular, proteincompositions such as bovine serum albumin (BSA), nonfat powdered milk,and gelatin are widely used with powdered milk being most preferred.

Labels

The particular label or detectable group used in the assay is not acritical aspect of the invention, as long as it does not significantlyinterfere with the specific binding of the antibody used in the assay.The detectable group can be any material having a detectable physical orchemical property. Such detectable labels have been well-developed inthe field of immunoassays and, in general, most any label useful in suchmethods can be applied to the present invention. Thus, a label is anycomposition detectable by spectroscopic, photochemical, biochemical,immunochemical, electrical, optical or chemical means. Useful labels inthe present invention include magnetic beads (e.g., Dynabeads™),fluorescent dyes (e.g., fluorescein isothiocyanate, Texas red,rhodamine, and the like), radiolabels (e.g., ³H, ¹²⁵I, ³⁵S, ¹⁴C, or³²P), enzymes (e.g., horse radish peroxidase, alkaline phosphatase andothers commonly used in an ELISA), and colorimetric labels such ascolloidal gold or colored glass or plastic (e.g., polystyrene,polypropylene, latex, etc.) beads.

The label may be coupled directly or indirectly to the desired componentof the assay according to methods well known in the art. As indicatedabove, a wide variety of labels may be used, with the choice of labeldepending on sensitivity required, ease of conjugation with thecompound, stability requirements, available instrumentation, anddisposal provisions.

Non-radioactive labels are often attached by indirect means. Generally,a ligand molecule (e.g., biotin) is covalently bound to the molecule.The ligand then binds to an anti-ligand (e.g., streptavidin) moleculewhich is either inherently detectable or covalently bound to a signalsystem, such as a detectable enzyme, a fluorescent compound, or achemiluminescent compound. A number of ligands and anti-ligands can beused in conjunction with the labeled, naturally occurring anti-ligands.Alternatively, any haptenic or antigenic compound can be used incombination with an antibody.

The molecules can also be conjugated directly to signal generatingcompounds, e.g., by conjugation with an enzyme or fluorophore. Enzymesof interest as labels will primarily be hydrolases, particularlyphosphatases, esterases and glycosidases, or oxidotases, particularlyperoxidases. Fluorescent compounds include fluorescein and itsderivatives, rhodamine and its derivatives, dansyl, umbelliferone, etc.Chemiluminescent compounds include luciferin, and2,3-dihydrophthalazinediones, e.g., luminol. For a review of variouslabeling or signal producing systems which may be used, see U.S. Pat.No. 4,391,904.

Means of detecting labels are well known to those of skill in the art.Thus, for example, where the label is a radioactive label, means fordetection include a scintillation counter or photographic film as inautoradiography. Where the label is a fluorescent label, it may bedetected by exciting the fluorochrome with the appropriate wavelength oflight and detecting the resulting fluorescence. The fluorescence may bedetected visually, by means of photographic film, by the use ofelectronic detectors such as charge coupled devices (CCDs) orphotomultipliers and the like. Similarly, enzymatic labels may bedetected by providing the appropriate substrates for the enzyme anddetecting the resulting reaction product. Finally simple colorimetriclabels may be detected simply by observing the color associated with thelabel. Thus, in various dipstick assays, conjugated gold often appearspink, while various conjugated beads appear the color of the bead.

Some assay formats do not require the use of labeled components. Forinstance, agglutination assays can be used to detect the presence of thetarget antibodies. In this case, antigen-coated particles areagglutinated by samples comprising the target antibodies. In thisformat, none of the components need be labeled and the presence of thetarget antibody is detected by simple visual inspection.

VI. Assays for Modulators of CENP-E

CENP-E is a plus end-directed microtubule motor that is required formitosis. The present invention provides for the first time biologicallyactive CENP-E. The activity of CENP-E can be assessed using a variety ofin vitro assays, e.g., microtubule gliding assays (see Example II) orATPase assays (Kodama et al., J. Biochem. 99: 1465-1472 (1986); Stewartet al., Proc. Nat'l Acad. Sci. USA 90: 5209-5213 (1993). Microtubuledepolymerization assays can also be used to examine CENP-E activity(Lombillo et al., J. Cell Biol. 128:107-115 (1995)).

In addition, CENP-E activity can be examined by comparing antibodydepletion of CENP-E or inhibition of CENP-E in vitro using culturedcells or egg extracts. Samples that have been depleted or inhibited arecompared to control samples that are not inhibited/depleted or that havebiologically CENP-E added back to the sample. Characteristics such asspindle assembly and metaphase arrest are used to compare the effect ofCENP-E inhibition or depletion.

Such assays can be used to test for the activity of CENP-E isolated fromendogenous sources or recombinant sources. Furthermore, such assays canbe used to test for modulators of CENP-E. Because the plus end-directedmicrotubule motor activity of CENP-E is essential for mitosis,inhibition of CENP-E can be used to control cell proliferation.

Candidate agents encompass numerous chemical classes, though typicallythey are organic molecules, preferably small organic compounds having amolecular weight of more than 100 and less than about 2,500 daltons.Candidate agents comprise functional groups necessary for structuralinteraction with proteins, particularly hydrogen bonding, or ionic(electrostatic) interactions and typically include at least an amine,carbonyl, hydroxyl, sulfonyl or carboxyl group, preferably at least twoof the functional chemical groups. The candidate agents often comprisecyclical carbon or heterocyclic structures and/or aromatic orpolyaromatic structures substituted with one or more of the abovefunctional groups. Candidate agents are also found among biomoleculesincluding peptides, saccharides, fatty acids, steroids, purines,pyrimidines, derivatives, structural analogs or combinations thereof.Candidate agents are obtained from a wide variety of sources includinglibraries of synthetic or natural compounds. For example, numerous meansare available for random and directed synthesis of a wide variety oforganic compounds and biomolecules, including expression of randomizedoligonucleotides. Alternatively, libraries of natural compounds in theform of bacterial, fungal, plant and animal extracts are available orreadily produced. Additionally, natural or synthetically producedlibraries and compounds are readily modified through conventionalchemical, physical and biochemical means. Known pharmacological agentsmay be subjected to directed or random chemical modifications, such asacylation, alkylation, esterification, amidification to producestructural analogs.

In an embodiment provided herein, the candidate bioactive agents areproteins. The protein may be made up of naturally occurring amino acidsand peptide bonds, or synthetic peptidomimetic structures. For example,homo-phenylalanine, citrulline and noreleucine are considered aminoacids for the purposes of the invention. “Amino acid” also includesimino acid residues such as proline and hydroxyproline. The side chainsmay be in either the (R) or the (S) configuration. In the preferredembodiment, the amino acids are in the (S) or L-configuration. Ifnon-naturally occurring side chains are used, non-amino acidsubstituents may be used, for example to prevent or retard in vivodegradations.

In another embodiment, the candidate bioactive agents are naturallyoccurring proteins or fragments of naturally occurring proteins. Thus,for example, cellular extracts containing proteins, or random ordirected digests of proteinaceous cellular extracts, may be used. In oneembodiment, the libraries are of bacterial, fungal, viral, and mammalianproteins, with the latter being preferred, and human proteins beingespecially preferred.

In one embodiment, the candidate agents are peptides of from about 5 toabout 30 amino acids, with from about 5 to about 20 amino acids beingpreferred, and from about 7 to about 15 being particularly preferred.The peptides may be digests of naturally occurring proteins as isoutlined above, or random peptides. By randomized or grammaticalequivalents herein is meant that each nucleic acid and peptide consistsof essentially random nucleotides and amino acids, respectively. Sincegenerally these random peptides (or nucleic acids, discussed below) arechemically synthesized, they may incorporate any nucleotide or aminoacid at any position. The synthetic process can be designed to generaterandomized proteins or nucleic acids, to allow the formation of all ormost of the possible combinations over the length of the sequence, thusforming a library of randomized candidate bioactive proteinaceousagents.

In one embodiment, the library is fully randomized, with no sequencepreferences or constants at any position. In a preferred embodiment, thelibrary is biased. That is, some positions within the sequence areeither held constant, or are selected from a limited number ofpossibilities. For example, in a preferred embodiment, the nucleotidesor amino acid residues are randomized within a defined class, forexample, of hydrophobic amino acids, hydrophilic residues, stericallybiased (either small or large) residues, towards the creation ofcysteines, for cross-linking, prolines for SH-3 domains, serines,threonines, tyrosines or histidines for phosphorylation sites, etc., orto purines, etc.

In another embodiment, the candidate agents are nucleic acids. Bynucleic acid or “oligonucleotide” or grammatical equivalents hereinmeans at least two nucleotides covalently linked together. A nucleicacid of the present invention will generally contain phosphodiesterbonds, although in some cases, as outlined below, nucleic acid analogsare included that may have alternate backbones, comprising, for example,phosphoramide (Beaucage et al., Tetrahedron 49(10):1925 (1993) andreferences therein; Letsinger, J. Org. Chem. 35:3800 (1970); Sprinzl etal., Eur. J. Biochem. 81:579 (1977); Letsinger et al., Nucl. Acids Res.14:3487 (1986); Sawai et al., Chem. Lett. 805 (1984), Letsinger et al.,J. Am. Chem. Soc. 110:4470 (1988); and Pauwels et al., Chemica Scripta26:141 91986)), phosphorothioate (Mag et al., Nucleic Acids Res. 19:1437(1991); and U.S. Pat. No. 5,644,048), phosphorodithioate (Briu et al.,J. Am. Chem. Soc. 111:2321 (1989), O-methylphosphoroamidite linkages(see Eckstein, Oligonucleotides and Analogues: A Practical Approach,Oxford University Press), and peptide nucleic acid backbones andlinkages (see Egholm, J. Am. Chem. Soc. 114:1895 (1992); Meier et al.,Chem. Int. Ed. Engl. 31:1008 (1992); Nielsen, Nature, 365:566 (1993);Carlsson et al., Nature 380:207 (1996), all of which are incorporated byreference). Other analog nucleic acids include those with positivebackbones (Denpcy et al., Proc. Natl. Acad. Sci. USA 92:6097 (1995);non-ionic backbones (U.S. Pat. Nos. 5,386,023, 5,637,684, 5,602,240,5,216,141 and 4,469,863; Kiedrowshi et al., Angew. Chem. Intl. Ed.English 30:423 (1991); Letsinger et al., J. Am. Chem. Soc. 110:4470(1988); Letsinger et al., Nucleoside & Nucleotide 13:1597 (1994);Chapters 2 and 3, ASC Symposium Series 580, Carbohydrate Modificationsin Antisense Research, Ed. Y. S. Sanghui and P. Dan Cook; Mesmaeker etal., Bioorganic & Medicinal Chem. Lett. 4:395 (1994); Jeffs et al., J.Biomolecular NMR 34:17 (1994); Tetrahedron Lett. 37:743 (1996)) andnon-ribose backbones, including those described in U.S. Pat. Nos.5,235,033 and 5,034,506, and Chapters 6 and 7, ASC Symposium Series 580,Carbohydrate Modifications in Antisense Research, Ed. Y. S. Sanghui andP. Dan Cook. Nucleic acids containing one or more carbocyclic sugars arealso included within the definition of nucleic acids (see Jenkins etal., Chem. Soc. Rev. (1995) pp. 169-176). Several nucleic acid analogsare described in Rawls, C & E News Jun. 2, 1997 page 35. All of thesereferences are hereby expressly incorporated by reference. Thesemodifications of the ribose-phosphate backbone may be done to facilitatethe addition of additional moieties such as labels, or to increase thestability and half-life of such molecules in physiological environments.In addition, mixtures of naturally occurring nucleic acids and analogscan be made. Alternatively, mixtures of different nucleic acid analogs,and mixtures of naturally occurring nucleic acids and analogs may bemade. The nucleic acids may be single stranded or double stranded, asspecified, or contain portions of both double stranded or singlestranded sequence. The nucleic acid may be DNA, both genomic and cDNA,RNA or a hybrid, where the nucleic acid contains any combination ofdeoxyribo- and ribo-nucleotides, and any combination of bases, includinguracil, adenine, thymine, cytosine, guanine, inosine, xanthine,hypoxanthine, isocytosine, isoguanine, etc.

As described above generally for proteins, nucleic acid candidate agentsmay be naturally occurring nucleic acids, random nucleic acids, orbiased random nucleic acids. For example, digests of procaryotic oreukaryotic genomes may be used as is outlined above for proteins.

In a preferred embodiment, the candidate bioactive agents are organicchemical moieties, a wide variety of which are available in theliterature.

In a preferred embodiment, the candidate agent is a small molecule. Thesmall molecule is preferably 4 kilodaltons (kd) or less. In anotherembodiment, the compound is less than 3 kd, 2 kd or 1 kd. In anotherembodiment the compound is less than 800 daltons (D), 500 D, 300 D or200 D. Alternatively, the small molecule is about 75 D to 100 D, oralternatively, 100 D to about 200 D.

The modulators that are identified herein may be useful as leadcompounds for therapeutics, bioagricultural compounds, or diagnostics. Atherapeutic as used herein refers to a compound which is believed to becapable of modulating CENP-E in vivo which can have treatmentapplication in both human and animal disease. Modulation of CENP-E wouldbe desirable in a number of conditions including but not limited to:abnormal stimulation of endothelial cells (e.g., atherosclerosis), solidand hematopoetic tumors and tumor metastasis, benign tumors, forexample, hemangiomas, acoustic neuromas, neurofibromas, vascularmalfunctions, abnormal wound healing, inflammatory and immune disorderssuch as Rheumatoid Arthritis, Bechet's disease, gout or gouty arthritis,abnormal angiogenesis accompanying: rheumatoid arthritis, psoriasis,diabetic retinopathy, and other ocular angiogenic diseases such as,macular degeneration, corneal graft rejection, corneal overgrowth,glaucoma, Osler Webber syndrome, cardiovascular diseases such ashypertension, cardiac ischemia and systolic and diastolic dysfunctionand fungal diseases such as aspergillosis, candidiasis and topicalfungal diseases such as tinea pedis.

A bioagricultural compound as used herein refers to a chemical orbiological compound that has utility in agriculture and functions tofoster food or fiber crop protection or yield improvement. For example,one such compound may serve as a herbicide to selectively control weeds,as a fungicide to control the spreading of plant diseases, as aninsecticide to ward off and destroy insect and mite pests. In addition,one such compound may demonstrate utility in seed treatment to improvethe growth environment of a germinating seed, seedling or young plant asa plant regulator or activator.

A diagnostic as used herein is a compound that assists in theidentification and characterization of a health or disease state inhumans or other animals. The diagnostic can be used in standard assaysas is known in the art.

The modulators can be applied to generally any cell type wherein CENP-Emodulation is desired, i.e., eukaryotic, single celled and multicelledorganisms, plant and animal, vertebrate, invertebrate and mammalian.

Modulators of CENP-E are tested using biologically active CENP-E,preferably biologically active XCENP-E. Modulation is tested using oneof the in vitro assays described above, e.g., ATPase, microtubulebinding and/or gliding, spindle assembly, and metaphase arrest. It isunderstood that any of the assays can be repeated or different types ofassays can be used on the same candidate agent to further characterizethe candidate agent as a CENP-E modulator. In particular, where morethan one candidate agent is used, the assay can be repeated usingindividual candidate agents. Moreover, where a candidate agent is a leadcompound, further assays can be performed to optimize results until itis established whether that compound or one similar thereto has thedesired effect.

As described above, CENP-E is also a useful diagnostic tool in vitro fordetermining when a cell is entering mitosis. Reversible inhibitors ofCENP-E can be used to synchronize cells in culture. Fungal homologues ofXCENP-E also provide a diagnostic tool for identifying fungalinfections.

The present invention also provides for kits for screening formodulators of CENP-E. Such kits can be prepared from readily availablematerials and reagents. For example, such kits can comprise any one ormore of the following materials: biologically active CENP-E, reactiontubes, and instructions for testing CENP-E activity. Preferably, the kitcontains biologically active XCENP-E. A wide variety of kits andcomponents can be prepared according to the present invention, dependingupon the intended user of the kit and the particular needs of the user.For example, the kit can be tailored for ATPase assays or microtubulegliding assays.

All publications and patent applications cited in this specification areherein incorporated by reference as if each individual publication orpatent application were specifically and individually indicated to beincorporated by reference.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it will be readily apparent to one of ordinary skill inthe art in light of the teachings of this invention that certain changesand modifications may be made thereto without departing from the spiritor scope of the appended claims.

EXAMPLES

The following examples are provided by way of illustration only and notby way of limitation. Those of skill in the art will readily recognize avariety of noncritical parameters that could be changed or modified toyield essentially similar results.

Methods

A. Isolation of XCENP-E cDNA and DNA Constructs

Fragments spanning nucleotides 1-1707 and 6376-8080 of human CENP-E cDNA(Yen, et al., Nature 359:536-539 (1992)) were used to screen a λgt1Oadult Xenopus ovary cDNA library (Rebagliati, et al., Cell 42:769-777(1985)), hybridizing at 42° C. according to Church & Gilbert (Church, etal., Proc. Natl. Acad. Sci. USA 81:1991-1995 (1984)). cDNA cloneshybridizing to both probes were isolated and used in combination toisolate overlapping cDNA clones spanning the intervening region. Thesequence of both cDNA strands was determined by a combination ofautomated cycle sequencing (Applied Biosystems, Perkin Elmer) and manualsequencing using Sequence version 2.0 (USB).

Overlapping regions of the various cDNA clones were often not absolutelyidentical, but displayed single base differences at multiple positions.One clone encoding the N-terminal region of the rod domain contained 27additional nucleotides relative to one other clone spanning that region.Single base differences between cDNA clones were attributed topolymorphisms present in the outbred psuedotetraploid source materialused to construct the cDNA library. The 27 nucleotide relative insertionmay be a polymorphism, or may represent an alternatively spliced XCENP-Eisoform. Overlapping sequence was compiled using MacVector software(Kodak Scientific Imaging Systems, Rochester, N.Y.).

B. Expression and Purification of XCENP-E Motor Domain

Recombinant CENP-E was prepared in order to test its activity in amicrotubule gliding assay. The recombinant XCENP-E was prepared as afusion protein inducible by IPTG, with a c-myc epitope tag.

First, an XCENP-E was cloned into an expression plasmid. The 5′untranslated region of XCENP-E was removed by PCR using two primers: SEQID NO:9 CATATGACCATGGCCGAGGGAGATGCAG and SEQ ID NO:10GTCAGGTCAGCAACATACACG. These primers were used to amplify the 5′ end ofthe X-CENP-E cDNA and introduce NdeI and NcoI sites adjacent to and atthe start codon, respectively. This PCR product was subcloned into pCRII(InVitrogen) and then joined at the NruI site with a portion of theXCENP-E cDNA, to reconstruct a motor domain encoding cDNA with analtered start codon.

An NcoI-XhoI fragment spanning nucleotides 143-1939 was excised from thereconstructed cDNA fragment and ligated into NcoI/XhoI cut pET23d(Novagen) to yield pET23dXCE. This plasmid was digested with BsrGI andXhoI, blunted with Klenow, and a Klenow blunted 60 bp HincII-EcoRIfragment from pBSKS+myc (gift from S. Michealis) was ligated to thedigested pET23dXCE backbone in the presence of BsrGI to bias orientationof the insert. The resulting plasmid, pET23dXCEMycHis, encodes aminoacids 1-473 of XCENP-E linked at the C-terminus to the followingsequence: SEQ ID NO:11 TVSISLGDLTMEQKLISEEDLNFEHHHHHH. The c-myc epitopeis underlined.

This plasmid was transformed into E. coli strain BL21 (DE3) pLysS. Aculture inoculated with a single colony was gown at 37° C. in a modifiedLB medium (10 g bactotryptone, 5 g yeast extract, 5 g NaCl, 2 g MgSO₄, 1g casaminoacids per liter, and 200 mg ampicillin per liter) to OD 600 of−1. The cultures were allowed to cool to room temperature and expressionof fusion protein was induced with 0.5 mM IPTG at room temperature.After induction, the cells were used immediately to prepare fusionprotein. Cells that were pelleted and stored at low temperatures priorto protein isolation gave low to no yield of active protein; due toCENP-E sensitivity to denaturation.

Cells were harvested 4 hours after induction, immediately resuspended inlysis buffer (50 mM Tris-HCl pH 7.5, 50 mM NaCl, 1 mM PMSF, 0.1 mM ATP),and lysed by 3 passages through a trench press. Insoluble debris wasremoved by a centrifugation at 35,000 rpm in Sorvall T647.5 rotor for 40minutes at 4° C. Soluble protein in the supernatant was bound in batchto 0.5 ml of NTI-agarose resin (Qiagen) for 15 minutes at 4° C. Theresin was placed in a column, washed with 5 ml of the lysis buffersupplemented with 20 mM Imidazole. XCENP-E fusion protein recovered byelution in lysis buffer containing 100 mM imidazole and 1 mM DTT. Atypical yield was 2 mg of soluble XCENP-E protein from 1 liter ofbacterial culture. Freshly prepared protein was used to assay motility.Incubation of bacterially expressed XCENP-E motor protein for longerthan 24 hours at 4° C. led to loss of motility.

C. Fusion Protein Expression, Antibody Production, and Immunoblotting

The tail and rod regions (see FIG. 1) of XCENP-E were used to makeantibodies to CENP-E. Antigens for α-XCENP-E_(TAIL) (aa 2396-2954) andα-XCENP-E_(ROD) (aa 826-1106) were produced in E. coli strain BL21 (DE3)pLysS as hexahistidine fusion proteins using the pRSETB expressionplasmid (InVitrogen). Following induction with IPTG for 4-16 hours,bacteria were pelleted, washed and lysed by rapid freeze thaw followedby sonication. Inclusion bodies containing the fusion proteins werepurified and solubilized in 8M urea, 0.1 M sodium phosphate pH 8.0.

α-XCENP-E_(ROD) fusion protein was further purified over Ni-NTA agarose(Qiagen) according to the manufacturer's instructions. α-XCENP-E_(TAIL)protein was isolated from preparative SDS-PAGE gels as described in(Harlow, et al., Antibodies, A Laboratory Manual: Cold Spring HarborLaboratory) (1988)). These antigens were used to raise polyclonalantibodies in rabbits.

For affinity purification, antigen was coupled to cyanogen bromideactivated Sepharose (Pharmacia) according to the manufacturersinstructions. Antibodies were purified (Harlow, et al., Antibodies, ALaboratory Manual (1988)), eluting with 0.2 M glycine pH 2.5. Antibodieswere dialyzed into 10 mM K-HEPES pH 7.8, 100 mM KC1, 1 mM MgCl₂ andconcentrated using prerinsed centricon spin concentrators (Amicon,Beverly, Mass.) or Nanospin filter concentrators (Gelman Sciences, AnnArbor, Mich.).

For immunoblots, cytoplasmic extract prepared from metaphase H arrestedXenopus eggs (Murray, in Methods in Cell Biology, pp. 581-605 (Kay &Peng, eds., (1991)) was resolved on a 4% polyacrylamide gel (˜50μg/lane), transferred to nitrocellulose and lanes individually probedwith affinity purified α-XCENP-E_(TAIL) or α-XCENP-E_(ROD).

For localization of XCENP-E in cultured Xenopus XTC cells, asynchronouscultures of XTC cells were fixed in methanol and simultaneously stainedwith mouse monoclonal anti-α-tubulin antibody and affinity purifiedrabbit α-XCENP-E_(TAIL) antibody. Chromatin was visualized by stainingwith DAPI. Selected cells at progressive stages of the cell cycle wereexamined on the blot: interphase, prophase, prometaphase, metaphase,anaphase, and telophase. Similar staining was observed using(x-XCENP-E_(ROD) antibody.

Immunoblots were prepared as follows: proteins resolved by SDS-PAGE,transferred to nitrocellulose, blocked with TBS 5% nonfat dried milk(NFDM), and probed with 2 μg/ml affinity purified antibody overnight inTBS containing 0.05% Tween (TBST) containing 5% NFDM. Primary antibodywas visualized using ¹²⁵I-Protein A (Amersham) followed byautoradiography. Occasionally, instead of ¹²⁵I-protein A, alkalinephosphatase conjugated goat anti-rabbit secondary antibody (Promega) wasused according to the manufacturers instructions. Quantitativephosphoimaging was performed using a Molecular Dynamics model 445 SIphosphorimager.

D. Spindle Assembly In Vitro

CSF-arrested extract is an Xenopus egg extract that is arrested inmetaphase using cytostatic factor. CSF-arrested extract was preparedfrom Xenopus eggs essentially as described in Murray, in Methods in CellBiology, pp. 581-605 (Kay & Peng, eds., 1991); Sawin, et al., J. CellBiol. 112:925-940 (1991)). 10 mg/ml rhodamine labelled bovine braintubulin (Hyman, et al., in Methods in Enzymology, pp. 478-485 (Vallee,ed., 1992)) was added at a 1 μl/300 μl of extract.

Localization of XCENP-E was examined on mitotic spindles assembled invitro. Tubulin, DAPI-stained chromatin, and α-XCENP-E_(TAIL) stainingwas examined. Metaphase spindles were assembled in vitro by cyclingCSF-arrested Xenopus egg extract containing Xenopus sperm chromatinthrough interphase and arresting at the following metaphase as described(Sawin, et al., J. Cell Biol. 112:925-940 (1991)). Rhodamine labelledtubulin was added to the extracts to visualize tubulin containingstructures. Spindles were sedimented onto coverslips and stained withaffinity purified α-XCENP-E_(TAIL) antibody, followed by FITC-conjugatedsecondary antibody and DAPI.

For immunodepletion of extract, 100 μg of affinity purified α-XCENP-Eantibody or non-immune rabbit IgG (Calbiochem, San Diego, Calif.) wasbound to 30 μl slurry of protein A Affiprep beads (BioRad, Hercules,Calif.) for 1 hour at 4° C. in CSF-XB (Murray, 1991). Beads weresedimented, unbound antibody removed, and serially washed with CSF-XB,CSF-XB containing 0.5 M NaCl, and CSF-XB containing leupeptin, pepstatinA, and chymostatic (10 μg/ml each), and cytochalasin B 10 μg/ml. 100 μlof CSF extract was added to the beads and incubated rocking for 1 hourat 4° C. After sedimenting beads, depleted extract was removed andstored on ice until use.

Demembranated sperm prepared as described (Newmeyer, et al., in Methodsin Cell Biology, pp. 607-634 (Kay & Peng, eds. (1991)) were added to aportion of the extract at 1-2×10⁵/ml, and exit from metaphase arrestinduced at room temperature by addition of CaCl₂ to 0.6-0.8 mM finalconcentration. Extracts were periodically monitored by fluorescencemicroscopic examination of 1 μl aliquots squashed under a coverslip(Murray, in Methods in Cell Biology, pp. 581-605 (Kay & Peng, eds.,1991)). At 80 minutes following exit from metaphase one half volume ofthe appropriate extract was added and the reaction incubated for anadditional 80-120 minutes.

M-phase structures accumulating in extracts were scored at 160-200minutes total elapsed time. Both mock depleted and XCENP-E depletedextracts frequently failed to exit interphase, or failed to remainarrested at the second metaphase, probably as a consequence ofexperimental manipulation. Immunoprecipitates were washed 3 times withCSF-XB containing protease inhibitors and 0.1% Triton X-100 and examinedby SDS-PAGE and Coomassie staining.

For Coomassie staining and α-XCENP-E_(ROD) blot of α-XCENP-Eimmunoprecipitates, immunoprecipitates were prepared from CSF-arrestedextract (˜10 mg total protein) using affinity purified α-XCENP-E_(TAIL)antibody, affinity purified (α-XCENP-E_(ROD) antibody, or non-immunerabbit IgG. Immunoprecipitates were gently washed three times with TBScontaining 0.1% Triton-X100. 80% of each precipitate was resolved bySDS-PAGE on a 5-15% gel and proteins visualized by staining withCoomassie brilliant blue.

For antibody addition experiments, purified anti-XCENP-E antibody ornon-immune rabbit IgG (Calbiochem) at 10 mg/ml was added to CSF-arrestedextract at a 1:20 dilution, followed by demembranated sperm nuclei andCaCl₂. 80 minutes later, when a half volume of CSF arrested extract wasadded, a proportional amount of the appropriate antibody was added aswell.

Representative structures formed in the presence of 0.5 mg/ml rabbit IgGand in the absence of added antibody, and in the presence of 0.5 mg/mlα-XCENP-E_(TAIL) antibody were examined. Rabbit IgG and α-XCENP-E_(TAIL)(both at 10 mg/ml) were added to CSF-arrested metaphase Xenopus eggextract at a 1:20 dilution along with Xenopus sperm. Extracts were thencycled through interphase. At 80 minutes into interphase (prophase) ahalf volume of metaphase arrested extract containing 0.5 mg/ml of theappropriate antibody was added. 80 minutes later structures were scoredand images collected.

Quantitation of structures formed in extract containing no antibody(n=138), extract containing 0.5 mg/ml non-immune rabbit IgG (n=132), andextract containing 0.5 mg/ml α-XCENP-E_(TAIL) (n=114) at 80 minutesafter exit from interphase were examined. Structures present in therespective extracts were examined and scored as belonging to one of fourcategories: bipolar spindles with chromatin aligned at the metaphaseplate; bipolar spindles with misaligned chromosomes; monopolar spindles,including radial asters, half spindles and chromosomes associated withmicrotubules with indeterminant organization; and or other, includingmultipolar structures and groups of chromosomes apparently unassociatedwith microtubules.

E. Immunofluorescence Microscopy

Extract containing mitotic spindles assembled in vitro was diluted 30-50fold in BRB80 (80 mM KPIPES, 6 mM MgCl₂, 1 mM EGTA) containing 0.5%Triton X-100 and 30% glycerol. Spindles were sedimented at roomtemperature onto a coverslip through a 3 ml cushion of BRB80 containing0.5% Triton X-100 and 40% glycerol at 7000 rpm in a Sorvall HS4 rotor.Coverslips were fixed in −20° C. methanol, rehydrated in TBS-Tx (150 mMNaCl, 20 mM Tris pH 7.6, 0.1% Triton X-100), blocked for 1 hour with 1%BSA in TBS-Tx and probed with 5 μg/ml affinity purified antibody in 1%BSA in TBS-Tx. After washing with TBS-Tx, primary antibody wasvisualized using by FITC-conjugated secondary goat anti-rabbit antibody(Cappel).

Xenopus XTC cells cultured on coverslips in 60% L15 medium containing10% fetal calf serum at room temperature in ambient atmosphere wererinsed in TBS, fixed in −20° C. methanol and stained with affinitypurified antibody as described above. Monoclonal anti-alpha tubulinantibody DM1A (Sigma) was used at a dilution of 1:1000 to stainmicrotubules.

Fluorescent images were collected using a Princeton Instruments cooledCCD mounted on a Zeiss Axioplan microscope controlled by Metamorphsoftware (Universal Imaging, West Chester, Pa.). Image processing wasperformed using both Metamorph and Adobe Photoshop software.

F. Preparation of Polarity Marked Microtubules and Motility Assay

Taxol stabilized microtubule seeds brightly labelled with rhodamine wereprepared by incubating a 1:1 ratio of rhodamine labelled bovine braintubulin (Hyman, et al., In Methods in Enzymology, pp. 478-485 (Vallee,ed., 1992)) with unlabelled bovine brain tubulin at a final tubulinconcentration of 2.5 mg/ml in PEM80 (80 mM Pipes pH 6.9, 1 mM EGTA, 1 mMMgCl₂) containing 10% glycerol, 1 μM taxol, 1 mM GTP at 37° C. for 15minutes. This mixture was then diluted with 2.75 volumes of warm PEM80containing 20 μM taxol and 2 mM GTP, and sheared by 5 passes through aHamilton syringe.

Dimly rhodamine-labelled extensions were grown from the brightlylabelled seeds in PEM80 containing 1 mM GTP and 1.5 mg/ml tubulincocktail consisting of a mixture of N-ethyl maleimide modified tubulin(Hyman, et al., in Methods in Enzymology, pp. 478-485 (Vallee, ed.,1992)), unlabelled tubulin and rhodamine labelled tubulin at a ratio of0.1/0.52/0.38 for 30 minutes at 37° C. The resulting suspension ofpolarity marked microtubules was diluted with PEM80 containing 10 μMtaxol and used to test motility.

25 μl flow chambers prepared from cover slips sealed with an Apiezongrease, were preadsorbed with a 1:10 diluted mouse ascities fluidcontaining anti-myc monoclonal antibody 9EI0 (Evans, et al., Mol. Cell.Biol. 5:3610-3616 (1985)), washed with 50 μl PEM80, incubated withXCENP-E motor protein diluted to 0.1 mg/ml, and unbound protein removedby rinsing with 50 μl of PEM80. A microtubule/ATP mix consistingpolarity marked microtubules in PEM80 containing 10 μM taxol, 2 mMMgATP, and an oxygen scavenging system (0.1 mg/ml catalase, 0.03 mg/mlglucose oxidase, 10 mM glucose, 0.1% β-mercaptoethanol (Kishino, et al.,Nature 33:74-76 (1989)) was then flowed into the chamber.

Movement of microtubules was monitored at room temperature on a ZeissAxioplan fluorescence microscope fitted with 63X Plan-Apochromat oilimmersion objective, and a Princeton instruments cooled CCD. Automatedtime-lapse image acquisition and data analysis was performed using theMetaMorph software package (Universal Imaging, West Chester, Pa.).

Example I Identification of Xenopus CENP-E

To investigate the role of CENP-E in mitotic spindle formation in vitrousing extracts of Xenopus eggs used low stringency hybridizationfollowed by library rescreening was used to clone the Xenopus homologueof CENP-E. This clone was subsequently used to raise antibodies suitablefor immunodepletion and antibody addition studies. The nucleotidesequence (SEQ ID NO:2) encodes a protein of 2954 amino acids with apredicted molecular mass of 340 kDa (SEQ ID NO:1, FIG. 1C). Thepredicted structure of Xenopus CENP-E (XCENP-E) is similar to humanCENP-E (hCENP-E), consisting of a 500 amino acid globular amino-terminaldomain containing a kinesin-like microtubule motor domain linked to aglobular tail domain by a region predicted to form a long, discontinuousα-helical coiled coil (Lupas, et al., Science 252, 1162-1164 (1991);Berger, et al., Proc. Natl. Acad. Sci. USA 92:8259-8263 (1995)) (FIG.1A). Within the core of the motor domain (residues 1-324) XCENP-E andhCENP-E share 74% identity, significantly greater than that sharedbetween XCENP-E and its nearest phylogenetic (evolutionary) neighbors(Moore, et al., Bioessays 18:207-219 (1996)). Outside the amino-terminaldomain lie three additional regions which share greater than 25%identity with human CENP-E, but not with other kinesin-like proteins(FIG. 1). On the basis of these regions of identity and its largepredicted size, the conclusion was made that XCENP-E is the Xenopushomologue of human CENP-E.

Example II XCENP-E is a Plus End-Directed Microtubule Motor

Both human and Xenopus CENP-E are localized to the centromeres ofmitotic chromosomes throughout all phases of chromosome movement. Thislocalization places CENP-E in a position to mediate attachment ofchromosomes to microtubules, movement of chromosomes during congression,and movement of chromosomes toward the spindle poles during anaphase A.

To test directly if CENP-E is a microtubule motor and to determine thedirectionality of CENP-E movement, the amino-terminal 473 amino acids ofXCENP-E, containing the kinesin-like motor domain, was fused at theC-terminus to 31 amino acids containing an 9 amino acid c-myc epitopetag followed by a hexahistidine tag (see FIG. 2A, and FIG. 1C,amino-terminal boxed region).

This protein was produced in E. coli, and purified over nickel-agarose,yielding the expected 57 kDa polypeptide as the major product (FIG. 2A,lane 1). Immunoblotting with a α-myc monoclonal antibody (9ElO) (Evans,et al., Mol. Cell. Biol. 5:3610-3616 (1985)) confirmed the 57 kDaprotein as the XCENP-E fusion protein (FIG. 2B, lane 2, arrowheads).

The XCENP-E fusion protein was tethered to a glass coverslip using theα-myc antibody and gliding of polarity marked microtubules containingbrightly fluorescent rhodamine labelled seeds near their minus ends(Howard, et al., in Motility Assays for Motor Proteins, pp. 105-113(Scholey, ed., 1993)) was recorded by time-lapse digital fluorescencemicroscopy. Representative time points demonstrating three examples ofplus end-directed movement are presented in FIG. 2B. Microtubules movedat a velocity of 5.1 μM/min±1.7 (n=49) with brightly fluorescent seedsleading, indicating that the immobilized XCENP-E fusion protein wasmoving toward microtubule plus ends. No movement was observed in theabsence of fusion protein. When assayed in the absence of α-myc antibodythe XCENP-E fusion protein also supported microtubule gliding, albeitless robustly.

This experiment demonstrates that CENP-E has plus-ended microtubulemotor activity. Furthermore, by perturbing CENP-E function in Xenopusegg extracts, as shown below in Examples III-V, it was shown thatcongression in vitro requires a kinetochore-associated microtubulemotor. This result contrasts with a prevailing model describing mitoticspindle formation in Xenopus egg extracts in vitro (Vernos, et al.,Trends in Cell Biol. 5:297-301 (1995); Heald, et al., Nature 382, 420-5(1996); Hyman, et al., Cell 84:401-410 (1996)). Both bipolar spindleswith misaligned chromosomes and monopolar structures were observed whenXCENP-E, a kinetochore-specific protein, was removed, or when XCENP-Efunction is impaired by addition of α-XCENP-E antibody (see ExamplesIII-V below). These findings indicate that during normal mitotic spindleformation, CENP-E plays an essential role in mitotic spindle assemblyand in prometaphase chromosome movements that result in metaphasechromosome alignment, via its activity as a plus-end directedmicrotubule motor activity.

Example III XCENP-E Associates with Xenopus Centromeres In Vivo and InVitro

To verify that Xenopus CENP-E exhibits a cell cycle-dependentkinetochore association, polyclonal antibodies were raised against tworecombinant antigens, one spanning the tail and C-terminal portion ofthe rod (α-XCENP-E_(TAIL), FIG. 1B) and the other corresponding to aportion of the N-terminus of the rod domain (α-XCENP-E_(ROD); FIG. 1B).

Immunoblotting of Xenopus egg extract reveals that the α-XCENP-E_(TAIL)antibody specifically recognizes XCENP-E as a single band of greaterthan 300 kDa. The α-XCENP-E_(ROD) antibody specifically recognizesXCENP-E and another protein of slightly lower molecular weight that maybe a distinct isoform of XCENP-E lacking the tail domain, or XCENP-Ethat has lost its tail domain as a result of partial proteolysis.

Immunostaining of cultured Xenopus XTC cells using α-XCENP-E_(TAIL)antibody revealed patterns of cell cycle-dependent localization similarto that observed for mammalian CENP-E (Yen, et al., Nature 359:536-539(1992); Brown, et al., J. Cell Sci. 109:961-969 (1996)) with theexception that during interphase XCENP-E was localized to the nucleus,consistent with the presence of a nuclear localization signal (Boulikas,et al., Gene Express. 3:193-227 1993)) at the C-terminal end of the roddomain (FIGS. 1A, NLS, and 1C underlined sequence, RKKTK). Nuclearstaining intensity was variable from cell to cell, probably reflectingdifferent levels of XCENP-E accumulation, as observed for cytoplasmicCENP-E staining of interphase human cells (Yen, et al., Nature359:536-539 (1992); Brown, et al., J. Cell. Biol. 125:1303:1312 (1994)).

Early in prometaphase XCENP-E localizes to discrete spots associatedwith condensed mitotic chromosomes. During metaphase and early anaphase,XCENP-E remains in discrete foci on chromosomes, and is also apparent atthe spindle poles. XCENP-E is found at the spindle midzone during lateanaphase and telophase. α-XCENP-E_(TAIL) immunostaining of metaphasespindles assembled using cytostatic factor (CSF)-arrested Xenopus eggextracts cycled through interphase (Murray, in Methods in Cell Biology,pp. 581-605 (Kay & Peng, eds. 1991); Sawin, et al., J. Cell Biol.112:925-940 (1991)) revealed that XCENP-E was also associated withkinetochores assembled in vitro. Similar patterns of staining wereobserved in XTC cells and on spindles assembled in vitro usingα-XCENP-E_(ROD) antibody.

Example IV XCENP-E is Required for Congression

To determine the aspect(s) of mitosis for which XCENP-E is required,α-XCENP-E_(TAIL) antibody (made to the tail domain of XCENP-E0 was usedto deplete XCENP-E from Xenopus egg extracts arrested in metaphase(CSF-extract). Immunoblotting of control and XCENP-E depletedCSF-extracts revealed that greater than 95% of XCENP-E could be removedby immunodepletion with this antibody. Unrelated antigens, such as XNuMA(Merdes, et al., Cell 87:447-458 (1996)), were unaffected by depletionof XCENP-E.

To examine the effects of immunodepletion on spindle assembly andchromosome movement, demembranated Xenopus sperm nuclei were added toundepleted, mock depleted and XCENP-E depleted CSF-extracts. Extractswere released from CSF-imposed metaphase arrest by addition of calciumand allowed to cycle through interphase and into the subsequent M-phase,whereupon an additional aliquot of the appropriate uncycled,metaphase-arrested XCENP-E depleted, mock depleted or undepleted extractwas added to re-impose a metaphase arrest, thus allowing theaccumulation of M-phase structures.

While mock depleted and undepleted extracts yielded predominantlybipolar spindles with chromosomes aligned at the metaphase plate,depletion of XCENP-E produced a five-fold increase in the number ofbipolar spindles with misaligned chromosomes, as well as smallerincrease in the percentage of monopolar structures, including radialasters, half spindles, and chromosomes associated with microtubules withindeterminate organization. Extended incubation failed to alter theproportion of bipolar spindles with properly aligned chromosomes. Thisfinding, and the presence of chromosomes resembling non-disjoinedmetaphase sister chromatids on structures formed in the absence ofXCENP-E indicates that depletion of XCENP-E prevents congression ofchromosomes to the metaphase plate despite apparently normal spindleassembly and chromosome attachment.

Three independent experiments revealed in every case a decrease in thepercentage of metaphase spindles accompanied by an increased percentageof bipolar/misaligned and monopolar structures, although thedistribution of the aberrant structures between the monopolar andbipolar/misaligned classes was variable. Failure of XCENP-E depletion tototally prevent the appearance of bipolar spindles with properly alignedchromosomes could be due to residual XCENP-E (below detection limit),may reflect the actions of other motor proteins functioning in partialredundancy with XCENP-E, or may simply reflect that proportion ofspindles in which the chromosomes were already sufficiently aligned.These data indicate that XCENP-E, or a complex containing XCENP-E, isrequired for chromosome congression.

To test the possibility that a multiprotein complex had been removed,the proteins immunodepleted by α-XCENP-E_(TAIL) were compared with thoseprecipitated by α-XCENP-E_(ROD) antibody. Examination of the proteinsimmunodepleted by α-XCENP-E_(TAIL) revealed the presence of multiplebands. This result was not surprising, given that XCENP-E is relativelylow in abundance compared to other spindle proteins such as NuMA (8-14μg/ml (Merdes, et al., Cell 87:447-458 (1996)), XKCM1 (10 μg/ml), andXklp2 (16 μg/ml, (Boleti, et al., J. Cell. Biol. 125:1303-1312).

Immunoprecipitates prepared with α-XCENP-E_(ROD) antibody also containedmultiple proteins, only two of which were obviously held in common withthe α-XCENP-E_(TAIL) immunoprecipitate. Immunoblotting ofα-XCENP-E_(TAIL) and α-XCENP-E_(ROD) immunoprecipitates withα-XCENP-E_(ROD) antibody revealed that one of the proteins is XCENP-E,and that the other protein of slightly lower molecular weight, is theadditional XCENP-E related protein shown earlier to be recognized inunmanipulated extract by the α-XCENP-E_(ROD) antibody. The presence ofthis XCENP-E-related protein in immunoprecipitates prepared using theα-XCENP-E_(TAIL) antibody, which does not directly recognize this lowermolecular weight species, provides evidence that like mostkinesin-related proteins, XCENP-E exists in a complex that is at leastdimeric.

Example V Addition of α-XCENP-E Antibody Disrupts Metaphase SpindleFormation

As a further test of the requirement of CENP-E in mediating chromosomecongression, especially in view of the removal of multiple proteins uponimmunodepletion of XCENP-E, XCENP-E function was perturbed in situ byaddition of the monospecific α-XCENP-E_(TAIL) antibody to CSF-arrestedXenopus egg extracts. These extracts were cycled through interphase andarrested at the subsequent M-phase.

As observed upon immunodepletion of XCENP-E, addition ofα-XCENP-E_(TAIL) antibody resulted in almost total elimination ofbipolar spindles with properly aligned chromosomes. This loss wasaccompanied by an increase in the percentage of bipolar spindles withmisaligned chromosomes, indicating a role for XCENP-E in congression.Also observed was a large increase in the proportion of monopolarstructures suggesting an additional role for XCENP-E in establishment ormaintenance of spindle bipolarity. Similar results were obtained in fourindependent experiments, and also using α-XCENP-E_(ROD) antibody.

The monopolar structures observed upon addition of α-XCENP-E antibodycould arise from disruption of bipolar spindle assembly. This sort ofspindle perturbation has also been observed following overexpression ofthe p5O subunit of dynactin, which also localizes to kinetochores(Echeverri, et al., J. Cell Biol. 132:617-633 (1996)). p5Ooverexpression disrupts spindle bipolarity, yielding two apparentmonopoles. On the other hand, monopoles may also arise from disruptionof sister chromatid cohesion upon entry into anaphase (Murray, et al.,Proc. Natl. Acad. Sci. USA 93:12327-12332 (1996)). Since apparentlynon-disjoined sister chromatids are visible in structures formed in thepresence of α-XCENP-E_(TAIL) antibody, the monopolar structures observedfor XCENP-E are unlikely to be the products of premature anaphase. Thesefindings support a role for XCENP-E during prometaphase in establishmentor maintenance of bipolarity, as well as in congression.

Consistent with an essential role for XCENP-E in chromosome movement,chromosomes associated with monopolar structures formed in the presenceof anti-XCENP-E antibody were often found distributed both at theperiphery and within the aster of microtubules. In contrast, the smallproportion of monopolar structures formed in control extractschromosomes were invariably localized at the periphery of the aster.Unlike perturbation of XKCM1, a relatively abundant Xenopus kinesinsuperfamily member, which induces formation of large asters as aconsequence of decreased microtubule catastrophe (Walczak, et al., Cell84:3747 (1996)), the asters formed in extracts to which α-XCENP-E_(TAIL)antibody was added, or from which XCENP-E had been removed, were notunusually large. This observation suggests that XCENP-E does not play arole in regulating microtubule dynamics analogous to that played byXKCM1.

1-42. (canceled)
 43. An antibody which specifically binds to an isolatedbiologically active centromere binding protein E (CENP-E), wherein theCENP-E (i) has plus end-directed microtubule motor activity, and (ii)comprises an amino acid sequence having at least 80% sequence identitywith amino acid residues 1-324 of SEQ ID NO:
 1. 44. The antibodyaccording to claim 43, wherein the antibody is a polyclonal antibody.45. The antibody according to claim 43, wherein the antibody is amonoclonal antibody.
 46. The antibody according to claim 43, wherein theantibody is humanized.
 47. The antibody according to claim 43, whereinthe antibody is a chimeric antibody.
 48. The antibody according to claim43, wherein the antibody is labeled.
 49. The antibody according to claim43, wherein the antibody inhibits at least one CENP-E activity uponbinding to CENP-E.
 50. The antibody according to claim 49, wherein theat least one CENP-E activity is plus end-directed microtubule motoractivity.
 51. The antibody according to claim 49, wherein the at leastone CENP-E activity is ATPase activity.
 52. The antibody according toclaim 49, wherein the at least one CENP-E activity is microtubulebinding activity.
 53. An antibody which specifically binds to anisolated polypeptide consisting of SEQ ID NO:
 1. 54. The antibodyaccording to claim 53, wherein the antibody is a polyclonal antibody.55. The antibody according to claim 53, wherein the antibody is amonoclonal antibody.
 56. The antibody according to claim 53, wherein theantibody is humanized.
 57. The antibody according to claim 53, whereinthe antibody is a chimeric antibody.
 58. The antibody according to claim53, wherein the antibody is labeled.
 59. The antibody according to claim53, wherein the antibody inhibits at least one CENP-E activity uponbinding to CENP-E.
 60. The antibody according to claim 59, wherein theat least one CENP-E activity is plus end-directed microtubule motoractivity.
 61. The antibody according to claim 59, wherein the at leastone CENP-E activity is ATPase activity.
 62. The antibody according toclaim 59, wherein the at least one CENP-E activity is microtubulebinding activity.
 63. An antibody which specifically binds to apolypeptide domain consisting of amino acids 826-1106 of SEQ ID NO: 1.64. The antibody according to claim 63, wherein the antibody is apolyclonal antibody.
 65. The antibody according to claim 63, wherein theantibody is a monoclonal antibody.
 66. The antibody according to claim63, wherein the antibody is humanized.
 67. The antibody according toclaim 63, wherein the antibody is a chimeric antibody.
 68. The antibodyaccording to claim 63, wherein the antibody is labeled.
 69. An antibodywhich specifically binds to a polypeptide domain consisting of aminoacids 826-1106 of SEQ ID NO:
 1. 70. The antibody according to claim 69,wherein the antibody is a polyclonal antibody.
 71. The antibodyaccording to claim 69, wherein the antibody is a monoclonal antibody.72. The antibody according to claim 69, wherein the antibody ishumanized.
 73. The antibody according to claim 69, wherein the antibodyis a chimeric antibody.
 74. The antibody according to claim 69, whereinthe antibody is labeled.